Targeted interferons demonstrating potent apoptotic and anti-tumor activities

ABSTRACT

Novel chimeric moieties that show significant efficacy against cancers are provided. In certain embodiments the chimeric moieties comprise a targeting moiety attached to an interferon. In certain embodiments, the chimeric moieties comprise fusion proteins where an antibody that specifically binds to a cancer marker is fused to interferon alpha (IFN-α) or interferon beta (IFN-β).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 12/985,122, filed onJan. 5, 2011, which is a Continuation of U.S. Ser. No. 12/650,329, filedon Dec. 30, 2009, now U.S. Pat. No. 8,258,263, issued on Sep. 4, 2012,which is a Continuation-in-Part of PCT/US2008/077074 (WO 2009/039409),filed on Sep. 19, 2008, which claims priority to and benefit of U.S.Ser. No. 60/994,717, filed on Sep. 21, 2007, all of which areincorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Grant No.CA087990, awarded by the National Institutes of Health. The Governmenthas rights in the invention.

FIELD OF THE INVENTION

This invention pertains to the field of oncology. Chimeric constructsare provided that have significant anti-cancer activity.

BACKGROUND OF THE INVENTION

Although spontaneous immune responses against tumor-associated antigens(TAAs) (Hrouda et al. (1999) Semin. Oncol. 26: 455-471) can be detected(Disis et al. (1997) J. Clin. Oncol. 15: 3363-3367), malignant cellscausing disease fail to elicit an immune response that leads torejection. Many studies have demonstrated that it is possible to enhancethe immunogenicity of tumor cells by introducing immunostimulatorymolecules such as cytokines and costimulatory molecules into them(Dranoff and Mulligan (1995) Adv. Immunol. 58: 417-454; Hrouda et al.(1999) Semin. Oncol. 26: 455-471; Hurford et al. (1995) Nat. Genet. 10:430-435); however, effective gene transfer still remains a challenge. Inaddition, eradication of residual cancer cells may require the targetingof widely scattered micrometastatic tumor deposits that are notaccessible to direct gene transfer.

Both the innate and the adaptive immune responses are essential forproviding protection against infectious pathogens and tumors. Thecross-talk between innate and adaptive immunity is regulated byinteractions between cells and cytokines. Cytokines produced by cells ofthe innate immune system can, directly or indirectly, activate the cellsof the adaptive immune response and can play an important role ineliciting protective antitumor immunity (Belardelli and Ferrantini(2002) Trends Immunol. 23: 201-208). Central to the activation of theinnate immune system is the detection of bacterial products or “danger”signals that lead to the release of proinflammatory cytokines, such asIFN-α, TNF-α, and IL-1.

IFN-α is a proinflammatory cytokine with potent antiviral andimmunomodulatory activities and is a stimulator of differentiation andactivity of dendritic cells (DCs) (Santini et al. (2000) J. Exp. Med.191: 1777-1788). Type I IFNs (IFN-α and IFN-β) have multiple effects onthe immune response (Theofilopoulos et al. (2005) Annu. Rev. Immunol.23: 307-336). IFN-α plays a role in the differentiation of Th1 cells(Finkelman et al. (1991) J. Exp. Med. 174: 1179-1188) and the long-termsurvival of CD8+ T cells in response to specific antigens (Tough et al.(1996) Science 272: 1947-1950).

Multiple studies have shown that IFNs are also capable of exertingantitumor effects in both animal models (Ferrantini et al. (1994) J.Immunol. 153: 4604-4615) and cancer patients (14. Gutterman et al.(1980) Ann. Intern. Med. 93: 399-406). In addition to enhancing theadaptive antitumor immune response, IFN-α can increase expression of thetumor suppressor gene P53 (Takaoka et al. (2003) Nature 424: 516-523),inhibit angiogenesis (Sidky and Borden (1987) Cancer Res. 47:5155-5161), and prime apoptosis (Rodriguez-Villanueva and McDonnell(1995) Int. J. Cancer 61: 110-11417) in tumor cells. Although theseproperties suggest that IFN-α should be an effective therapeutic for thetreatment of cancer, its short half-life and systemic toxicity havelimited its usage.

SUMMARY OF THE INVENTION

In various embodiments this invention pertains to the discovery thatattaching an interferon to a targeting moiety (e.g., a molecule thatspecifically and/or preferentially binds a marker on or associated witha cell) substantially improves the therapeutic efficacy of theinterferon and appears to reduce systemic toxicity. Accordingly, invarious embodiments, this invention provides constructs comprising aninterferon attached to a targeting moiety and uses of such constructs tospecifically and/or preferentially inhibit the growth or proliferationor even to kill certain target cells (e.g., cancer cells).

Accordingly, in certain embodiments, a chimeric construct is providedwhere the construct comprises an interferon (e.g., interferon-alpha,interferon-beta, interferon-gamma, etc.) attached to a targeting moietythat binds to a tumor associated antigen (TAA), where the construct whencontacted to a tumor cell results in the killing or inhibition of growthor proliferation of the tumor cell. In certain embodiments a chimericconstruct is provided where the construct comprises an interferonattached to a targeting moiety that binds to a cell surface marker or acell-associated marker, where the targeting is not attached to theinterferon by a (Gly₄Ser)₃ (SEQ ID NO:5) linker. In various embodimentsthe interferon is a type 1 interferon. In various embodiments theinterferon is a type 2 interferon. In various embodiments the interferonis an interferon alpha, an interferon-beta, or an interferon-gamma. Incertain embodiments the targeting moiety is an antibody that binds atumor associated antigen. In certain embodiments the targeting moiety ischemically coupled to the interferon. In certain embodiments thetargeting moiety is joined to the interferon with a peptide linker. Incertain embodiments the peptide linker is fewer than 15, fewer than 14,fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8,fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, orfewer than 2 amino acids in length. In certain embodiments the linker is15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid inlength. In certain embodiments the linker is not (Gly₄Ser)₃ (SEQ IDNO:5). In certain embodiments the linker is a linker that is resistantor substantially resistant to proteolysis. In certain embodiments thepeptide linker is Gly₄Ser (SEQ ID NO:6). In certain embodiments thelinker comprises or consists of an amino acid sequence found in Table 4.In certain embodiments the construct is a recombinantly expressed fusionprotein. In certain embodiments the antibody specifically binds a markerselected from the group consisting of EGFR, HER4, HER3, HER2/neu, MUC-1,G250, mesothelin, gp100, tyrosinase, and MAGE. In certain embodimentsthe targeting moiety is an antibody that binds CD20. In certainembodiments the targeting moiety is a single chain antibody thatcomprises the CDRS and/or the variable regions from an antibody selectedfrom the group consisting of anti-CD20 (rituximab), Ibritumomabtiuxetan, tositumomab, AME-133v, OCRELIZUMAB, OFATUMUMAB, TRU-015,IMMU-106, and the like. In various embodiments the targeting moiety isan antibody that binds HER2. In certain embodiments the antibody is a C6antibody. In certain embodiments the antibody comprises the VH and VLCDRs or VH and VL domains of C6 MH3-B1. In various embodiments theantibody is an IgG (e.g., IgG1, IgG3, etc.), an IgE, a single chain Fv(scFv), a FAB, a (Fab′)₂, an (ScFv)₂, and the like. In certainembodiments the antibody is an antibody selected form the groupconsisting of RITUXAN®, IF5, B1, 1H4, CD19, B4, B43, FVS191, hLL2, LL2,RFB4, M195, HuM195, AT13/5, HERCEPTIN®, 4D5, HuCC49, HUCC39ΔCH2 B72.3,12C10, IG5, H23, BM-2, BM-7, 12H12, MAM-6, and HMFG-1. In certainembodiments the antibody is an antibody that binds a member of the EGFreceptor family. In certain embodiments the antibody is selected fromthe group consisting of C6.5, C6ML3-9, C6 MH3-B1, C6-B1D2, F5, HER3.A5,HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12, EGFR.C10,EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8, HER4.B6, HER4.D4,HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8 and HER4.C7. Incertain embodiments the construct comprises an anti-HER2 IgG1 antibodyattached to an interferon.

Also provided are pharmaceutical formulations. In various embodimentsthe formulations comprise a chimeric construct comprising an interferonattached to a targeting moiety. In certain embodiments the chimericconstruct comprises a construct as described above (and/or herein below)(e.g., an anti-CD20-Interferon, and anti-HER2-interferon, etc.). Incertain embodiments the formulation is a unit dosage formulation. Incertain embodiments the formulation is a formulated for parenteraladministration. In certain embodiments the formulation is a formulatedfor administration via a route selected from the group consisting oforal administration, intravenous administration, intramuscularadministration, direct tumor administration, inhalation, rectaladministration, vaginal administration, transdermal administration, andsubcutaneous depot administration.

In various embodiments methods are provided for inhibiting growth and/orproliferation of a cancer cell. The methods typically involve contactingthe cancer cell with a chimeric construct as described herein. Incertain embodiments the cancer cell is a metastatic cell, and/or a cellis in a solid tumor. In certain embodiments the cancer cell is a breastcancer cell. In certain embodiments the cancer cell is a B celllymphoma. In certain embodiments the cancer cell is cell produced by acancer selected from the group consisting of a B cell lymphoma, lungcancer, a bronchus cancer, a colorectal cancer, a prostate cancer, abreast cancer, a pancreas cancer, a stomach cancer, an ovarian cancer, aurinary bladder cancer, a brain or central nervous system cancer, aperipheral nervous system cancer, an esophageal cancer, a cervicalcancer, a melanoma, a uterine or endometrial cancer, a cancer of theoral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tractcancer, a small bowel or appendix cancer, a salivary gland cancer, athyroid gland cancer, a adrenal gland cancer, an osteosarcoma, achondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibroushistiocytoma. In various embodiments the contacting comprisessystemically administering the chimeric moiety to a mammal. In certainembodiments the contacting comprises administering the chimeric moietydirectly into a tumor site. In certain embodiments the contactingcomprises intravenous administration of the chimeric moiety. In certainembodiments the cancer cell is a cancer cell in a human or in anon-human mammal.

In certain embodiments nucleic acids are provided that encode thechimeric constructs described herein. In various embodiments the nucleicacid encodes a fusion protein comprising an interferon attached to ananti-EGFR family member antibody, an anti-HER2 antibody, an anti-C6single-chain antibody, or to an anti-CD20 single chain antibody. Invarious embodiments the interferon encoded by the nucleic acid is a typeI interferon. In certain embodiments the interferon is IFN-α orinterferon-β. In various embodiments the nucleic acid encodes anantibody that comprises the VH and VL CDRs of C6MH3-B1. In variousembodiments nucleic acid encodes a peptide linker (e.g., as describedherein) attaching the antibody to the interferon. In certain embodimentsthe nucleic acid encodes the CDRs and/or the variable regions foranti-CD20 (rituximab).

Also provided is a cell comprising a nucleic acid as described above,that encodes a chimeric construct. In certain embodiments the cellexpresses the chimeric construct.

In various embodiments this invention provides the use of a chimericconstruct as described herein in the manufacture of a medicament toinhibit the growth and/or proliferation of a cancer cell.

In certain embodiments, the methods and constructs of this inventionspecifically exclude constructs using any of the antibodies disclosed inU.S. Patent Publication No: US 2002/0193569 A1. In certain embodimentsthe methods and constructs of this invention specifically excludeconstructs incorporating an anti-CD20 antibody. In certain embodimentsthe methods and constructs of this invention specifically excludeconstructs incorporating antibodies that bind to any of the followingtargets: CD19, CD20, CD22, CD33, CD38, EGF-R, HM1.24, phosphatidylserine antigen, HER-2, TAG-72, and/or MUC-1. In certain embodiments theconstructs described herein can be used in the treatment of pathologiessuch as multiple sclerosis, HCV mediated vasculitis, and the like.

DEFINITIONS

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide. Preferred“peptides”, “polypeptides”, and “proteins” are chains of amino acidswhose alpha carbons are linked through peptide bonds. The terminal aminoacid at one end of the chain (amino terminal) therefore has a free aminogroup, while the terminal amino acid at the other end of the chain(carboxy terminal) has a free carboxyl group. As used herein, the term“amino terminus” (abbreviated N-terminus) refers to the free α-aminogroup on an amino acid at the amino terminal of a peptide or to theα-amino group (imino group when participating in a peptide bond) of anamino acid at any other location within the peptide. Similarly, the term“carboxy terminus” refers to the free carboxyl group on the carboxyterminus of a peptide or the carboxyl group of an amino acid at anyother location within the peptide. Peptides also include essentially anypolyamino acid including, but not limited to peptide mimetics such asamino acids joined by an ether as opposed to an amide bond.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these regions of thelight and heavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases orexpressed de novo. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′₂, a dimerof Fab which itself is a light chain joined to V_(H)-C_(H)1 by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region (see, Fundamental Immunology, W. E.Paul, ed., Raven Press, N.Y. (1993), for a more detailed description ofother antibody fragments). While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such Fab′ fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein also includes antibody fragments eitherproduced by the modification of whole antibodies or synthesized de novousing recombinant DNA methodologies, including, but are not limited to,Fab′₂, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, unibodies, anddiabodies. In various embodiments preferred antibodies include, but arenot limited to Fab′₂, IgG, IgM, IgA, IgE, and single chain antibodies,more preferably single chain Fv (scFv) antibodies in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide.

In certain embodiments antibodies and fragments used in the constructsof the present invention can be bispecific. Bispecific antibodies orfragments can be of several configurations. For example, bispecificantibodies may resemble single antibodies (or antibody fragments) buthave two different antigen binding sites (variable regions). In variousembodiments bispecific antibodies can be produced by chemical techniques(Kranz et al. (1981) Proc. Natl. Acad. Sci., USA, 78: 5807), by“polydoma” techniques (see, e.g., U.S. Pat. No. 4,474,893), or byrecombinant DNA techniques. In certain embodiments bispecific antibodiesof the present invention can have binding specificities for at least twodifferent epitopes at least one of which is a tumor associate antigen.In various embodiments the antibodies and fragments can also beheteroantibodies. Heteroantibodies are two or more antibodies, orantibody binding fragments (e.g., Fab) linked together, each antibody orfragment having a different specificity.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

The term “interferon” refers to a full-length interferon or to aninterferon fragment (truncated interferon) or interferon mutant, thatsubstantially retains the biological activity of the full lengthwild-type interferon (e.g., retains at least 80%, preferably at least90%, more preferably at least 95%, 98%, or 99% of the full-lengthinterferon). Interferons include type I interferons (e.g.,interferon-alpha and interferon-beta) as well as type II inteferons(e.g., interferon-gamma). The interferon (e.g., IFN-α) can be fromessentially any mammalian species. In certain preferred embodiments, theinterferon is from a species selected from the group consisting ofhuman, equine, bovine, rodent, porcine, lagomorph, feline, canine,murine, caprine, ovine, a non-human primate, and the like. In variousembodiments the mutated interferon comprises one or more amino acidsubstitutions, insertions, and/or deletions.

An anti-HER2/neu antibody is an antibody that specifically orpreferentially binds a HER2/neu receptor.

As used herein, the term “subject” refers to a human or non-humananimal, including, but not limited to, a cat, dog, horse, pig, cow,sheep, goat, rabbit, mouse, rat, or monkey.

The term “C6 antibody”, as used herein refers to antibodies derived fromC6.5 whose sequence is expressly provided, for example, in U.S. Pat.Nos. 6,512,097 and 5,977,322, and in PCT Publication WO 97/00271. C6antibodies preferably have a binding affinity of about 1.6×10⁻⁸ orbetter for HER2/neu. In certain embodiments C6 antibodies are derived byscreening (for affinity to c-erbB-2/HER2/neu) a phage display library inwhich a known C6 variable heavy (V_(H)) chain is expressed incombination with a multiplicity of variable light (V_(L)) chains orconversely a known C6 variable light chain is expressed in combinationwith a multiplicity of variable heavy (V_(H)) chains. C6 antibodies alsoinclude those antibodies produced by the introduction of mutations intothe variable heavy or variable light complementarity determining regions(CDR1, CDR2 or CDR3), e.g., as described in U.S. Pat. Nos. 6,512,097 and5,977,322, and in PCT Publication WO 97/00271. In addition, C6antibodies include those antibodies produced by any combination of thesemodification methods as applied to C6.5 and its derivatives.

An “anti-EGFR family antibody” refers to an antibody that specificallybinds to a member of the epidermal growth factor receptor family (e.g.,an antibody that binds to ErbB-1, also named epidermal growth factorreceptor (EGFR), ErbB-2, also named HER2 in humans and neu in rodents,ErbB-3, also named HER3, and/or to ErbB-4, also named HER4).Illustrative anti-EGFR family antibodies include, but are not limited toantibodies such as C6.5, C6ML3-9, C6 MH3-B1, C6-B1D2, F5, HER3.A5,HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12, EGFR.C10,EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8, HER4.B6, HER4.D4,HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8 and HER4.C7 andthe like (see, e.g., U.S. Patent publications US 2006/0099205 A1 and US2004/0071696 A1 which are incorporated herein by reference).

A single chain FIT (“sFv” or “scFv”) polypeptide is a covalently linkedV_(H):V_(L) heterodimer which, in certain embodiments, may be expressedfrom a nucleic acid including V_(H)- and V_(L)-encoding sequences eitherjoined directly or joined by a peptide-encoding linker. Huston, et al.Proc. Nat. Acad. Sci. USA, 85: 5879-5883 (1988). A number of structuresfor converting the naturally aggregated, but chemically separated lightand heavy polypeptide chains from an antibody V region into an sFvmolecule that will fold into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site. See, e.g. U.S. Pat.Nos. 5,091,513 and 5,132,405, and 4,956,778.

“CD20” is a non-glycosylated phosphoprotein expressed on the surface ofmature B-cells (see, e.g., Cragg et al. (2005) Curr. Dir. Autoimmun., 8:140-174). It is also found on B-cell lymphomas, hairy cell leukemia,B-cell chronic lymphocytic leukemia on skin/melanoma cancer stem cells,and the like.

The phrase “inhibition of growth and/or proliferation” of a cancer cellrefers to decrease in the growth rate and/or proliferation rate of acancer cell. In certain embodiments this includes death of a cancer cell(e.g. via apoptosis). In certain embodiments this term also refers toinhibiting the growth and/or proliferation of a solid tumor and/orinducing tumor size reduction or elimination of the tumor.

The term “cancer marker” refers to biomolecules such as proteins,carbohydrates, glycoproteins, and the like that are exclusively orpreferentially or differentially expressed on a cancer cell and/or arefound in association with a cancer cell and thereby provide targetspreferential or specific to the cancer. In various embodiments thepreferential expression can be preferential expression as compared toany other cell in the organism, or preferential expression within aparticular area of the organism (e.g. within a particular organ ortissue).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate nucleic acid and amino acid sequences forvarious constructs described herein. FIG. 1A shows amino acid sequencesfor anti-HER2/neu IgG3 heavy chain-IFNα (SEQ ID NO:1) and anti-HER2/neuIgG3 light chain (SEQ ID NO:2). Single underline is linker, doubleunderline is murine IFN-α, no underline is anti-HER2/neu. FIG. 1B showsa nucleic acid sequence (SEQ ID NO:3) and an amino acid sequence (SEQ IDNO:4) for anti-CD20-IgG3-huIFNα with a Gly₄Ser (SEQ ID NO:6) linker. Itwill be appreciated that while the constructs in this figure are shownwith particular targeting moieties, particular linkers, and particularinterferons, in certain embodiments other targeting moieties, linkers,and interferons can be substituted therefore as described herein.

FIGS. 2A, 2B, 2C, and 2D illustrate the construction andcharacterization of anti-HER2/neu IgG3-IFN-α. FIG. 2A: Schematic diagramof anti-HER2/neu-IgG3-IFN-α. Solid areas represent anti-HER2/neuvariable regions. Open areas represent human IgG3 and κ constantregions. White circle regions represent murine IFN-α. FIG. 2B: SDS-PAGEof purified anti-HER2/neu-IgG3 (lanes 1 and 4), IgG3-IFN-α (lanes 2 and5), and anti-HER2/neu-IgG3-IFN-α (lanes 3 and 6) under nonreducing(lanes 1-3) or reducing (lanes 4-6) conditions. The molecular massmarker proteins are shown at the left of each gel. FIG. 2C:Anti-HER2/neu-IgG3 and anti-HER2/neu-IgG3-IFN-α bind HER2/neu.CT26/HER2, a murine colonic cell line expressing high levels of humanHER2/neu, was reacted with anti-HER2/neu-IgG3, IgG3-IFN-α, oranti-HER2/neu-IgG3-IFN-α with or without heparin followed by PE-labeledrabbit anti-human IgG. Dashed lines represent signal from cells withoutaddition of recombinant protein. FIG. 2D: The protective activity of theIFN-α standard and different IFN-α fusion proteins against VSV.Dilutions of 1 U of IFN-α standard, 0.21 ng (10 pM) ofanti-HER2/neu-IgG3-IFN-α, 0.21 ng (10 pM) of IgG3-IFN-α, or 0.17 ng (10pM) of anti-HER2/neu-IgG3 in 100 μl were prepared and added to L-929cells. After a 24-h incubation, 4000 PFU of VSV were added. Forty-eighthours later, viable cells were stained with crystal violet dye,dissolved by methanol, and solubilized dye was detected using an ELISAreader at 570 nm.

FIGS. 3A and 3B show in vivo antitumor activity of different IFN-αfusion proteins and rIFN-α. C3H/HeN mice were s.c. challenged with 1×10³38C13/HER2 cells and i.p. treated with either 2.5 μg (FIG. 3A) or 1 μg(FIG. 3B) of the indicated proteins at days 1, 3, and 5 after tumorchallenge. The tumor volume of each mouse is measured. Animals wereobserved until the diameter of the s.c. tumor reached 15 mm.

FIGS. 4A and 4B show that fusion of IgG3 to IFN-α improved its antitumoractivity and increased its in vivo half-life. FIG. 4A: Mice were treatedwith 9600 U of rIFN-α or 9600 U (4 μg) of IgG3-IFN-α at days 1 and 3after tumor challenge. Animals were followed for survival and sacrificedwhen the diameter of the s.c. tumor reached 15 mm. FIG. 4B: Groups ofthree C3H/HeN mice were injected i.p. with 66 μCi of ¹²⁵I-labeledrIFN-α, IgG3-IFN-α or anti-HER2/neu-IgG3-IFN-α. At various intervalsafter injection of the ¹²⁵I-labeled proteins residual radioactivity wasmeasured using a mouse whole body counter. The results represent themean of three mice. Bars, SD.

FIGS. 5A, 5B, 5C, and 5D show that IFN-α fusion proteins inhibited cellproliferation and induced apoptosis in 38C13/HER2 cells in vitro. IFN-αfusion proteins inhibited tumor cell proliferation. After incubation for48 h with different doses of the different fusion proteins, viable38C13/HER2 (FIG. 5A) or 38C13 (FIG. 5B) cells were measured using theMTS assay. These experiments were performed three times in triplicate;error bars, SD of the measurements. FIG. 5C: IFN-α fusion proteinsinduce apoptosis in 38C13/HER2 cells. In brief, 1×10⁶ 38C13/HER2 cellswere incubated with 1 nM of the indicated proteins for 72 h. The cellswere then washed, stained with Alexa Fluor 488, annexin V, and PI andwere analyzed by flow cytometry. The percentage of cells located in eachquadrant is indicated at the corner. FIG. 5D: IFN-α fusion proteinsinhibited proliferation of surviving 38C13/HER2 cells. In brief, 1×10⁶38C13/HER2 cells were labeled with 2.5 μM CFSE and immediately fixed(dash line), or treated with PBS (thin black line), or 1 nM of eitheranti-HER2/neu IgG3 (thin black line, overlaps with PBS control),IgG3-IFN-α (thick black line), or anti-HER2/neu-IgG3-IFN-α (black area)for 48 h. The cells were then washed and analyzed by flow cytometry. Thehistogram was obtained by gating on the population of live cells.

FIGS. 6A, 6B, and 6C show that IFN-α fusion proteins induced STAT1activation in 38C13/HER2 cells. In brief, 1×10⁷ 38C13/HER2 cells weretreated with 1000 U/ml of either anti-HER2/neu-IgG3-IFN-α (FIG. 6A) orIgG3-IFN-α (FIG. 6B) for the indicated times. The cell lysates wereseparated by SDS-PAGE and analyzed by Western blot using a polyclonalrabbit anti-phosphoSTAT1. To confirm equal loading of protein samples,blots were probed with a HRPconjugated rabbit polyclonal Ab againstGAPDH. FIG. 6C: The intensity of antiphosphoSTAT1 was normalized withthe intensity of anti-GAPDH for each indicated time point, and thevalues obtained were divided by the value at time 0 to obtain the foldactivation for STAT1. These experiments were performed twice; errorbars, SD of the measurements.

, Only point where the two groups differ with a p<0.05.

FIG. 7 IFN-α fusion proteins inhibit the growth of established tumor.C3H/HeN mice were injected s.c. with 1×10³ 38C13/HER2 cells. After 12days, mice were treated i.p. with 5 μg of the indicated protein for 3consecutive days. The tumor volume of each mouse is measured. Animalswere sacrificed when the diameter of the s.c. tumor reached 15 mm.

FIG. 8 shows binding of recombinant antibodies to human cells expressingCD20. Daudi cells were incubated with either recombinant IgG3 orrituximab followed by biotinylated rat anti-human IgG and PE-labeledstrepavidin and analyzed by flow-cytometry. A, cells with only thesecondary antibody; B, cells with recombinant IgG3; C, cells withrituximab.

FIG. 9 shows a diagram of the heavy chain of the antibody-IFN-α fusionprotein. In particular, the figure illustrates shortening of the(Gly₄Ser)₃ (SEQ ID NO:5) to a Gly₄Ser (SEQ ID NO:6) linker enablesproduction of full-length αCD20-IgG3-mIFNα.

FIG. 10 shows SDS-PAGE analysis of fractions eluted from protein ASepharose. Culture supernatants from cells expressinganti-CD-20-IgG3-IFNα with the (Gly₄Ser)₃ (SEQ ID NO:5) linker werepassed through the protein A Sepharose and the fusion protein boundprior to elution. A. Proteins were run without reduction. Lane 1, IgG3;Lanes 2-6, fractions eluted from protein A Sepharose. B. Proteins werereduced prior to analysis. Lane 2, IgG3; Lanes 3-7, fractions elutedfrom protein A Sepharose.

FIG. 11 shows SDS-PAGE analysis of proteins made by transient expressionin HEK293T cells. Lane 1, anti-CD20-IgG3-huIFNα with extended (Gly₄Ser)₃(SEQ ID NO:5) linker; Lane 2, anti-CD20-IgG3 huIFNα with shortenedGly₄Ser (SEQ ID NO:6) linker; Lane 3, anti-CD20-IgG3-muIFNα withextended (Gly₄Ser)₃ (SEQ ID NO:5) linker; Lane 4, anti-CD20-IgG3-muIFNαwith shortened Gly₃Ser linker; Lane 5, anti-CD20 IgG3.

FIG. 12 was shows an analysis of protein binding to Daudi cells usingFLOW cytometry. 1×10⁶ Daudi cells were stained with 1 μg of fusionprotein containing human IFN-α or RITUXAN®.

FIG. 13 shows an analysis of protein binding to 38C13/CD20 by FLOWcytometry.

FIG. 14. Daudi cells were incubated with various concentrations ofIFN-α, antibody or fusion protein for 72 hrs. Growth inhibition wasassessed using the CellTiter 96 AQueous cell proliferation assay.

FIG. 15. Daudi cells were treated with 10 pM of the indicated proteinsfor 72 hours. Cell viability and apoptosis was determined followingstaining with Annexin V and PI and analysis by FLOW cytometry.

FIG. 16. 38C13/CD20 cells were treated with 10 pM of the indicatedproteins for 48 hours. Cell viability and apoptosis was determinedfollowing staining with Annexin V and PI and analysis by FLOW cytometry.

FIG. 17 shows inhibition of cell proliferation following treatment withdifferent proteins at varying concentrations. 38C13-CD20 cells weretreated with the indicated proteins at varying concentrations for 48hours. After treatment the extent of proliferation was monitored usingthe MTS assay.

FIG. 18. 38C13/CD20 cells were treated with the different concentrationsof the indicated proteins for 48 hours. Cell viability and apoptosis wasdetermined following staining with Annexin V and PI and analysis by FLOWcytometry.

FIG. 19. Daudi cells were incubated for 72 hours with differentconcentrations of the fusion protein. Cell viability and apoptosis wasdetermined following staining with Annexin V and PI and analysis by FLOWcytometry.

FIG. 20. Daudi cells were treated for 72 hours with variousconcentrations of fusion proteins. MTS solution was added to quantitatecell viability.

FIG. 21. Daudi cells were incubated for 72 hours with 1 pM ofanti-CD20-IgG3-hIFNα with the Gly₄Ser linker (6) (Gly-Ser Linker) orwith 1 pM of anti-CD20-IgG3-hIFNα with the alpha helical linker (Alphahelix Linker). Cell viability and apoptosis was determined followingstaining with Annexin V and PI and analysis by FLOW cytometry.

FIG. 22 shows survival of mice inoculated with 5000 38C13-CD20 cells andtreated on days 1, 2 and 3 with HBSS or the indicated amounts of theanti-CD20-IFN-α fusion proteins.

FIG. 23 shows survival of mice inoculated with 5000 38C13-CD20 cells andtreated on days 5, 6 and 7 with 10 μg of anti-CD20-IgG1 (CD20-IgG1),anti-CD20-IgG3 (CD20-IgG3), rituximab or anti-CD20-IgG3-mIFNα(CD20-mIFNα) or HBSS.

FIG. 24 shows survival of mice inoculated with 5000 38C13-CD20 cells andtreated on days 5, 6 and 7 with 10 μg of anti-CD20-IgG3 (IgG3),anti-CD20-IgG3+IFNα (IgG3+IFNα), anti-DNS-IgG3-mIFNα (DNS-IFNα),anti-CD20-IgG3-mIFNα (CD20-IFNα) or HBSS.

FIG. 25. Groups of eight mice were injected with 5000 38C13-CD20 cellson day 0. On days 8, 9 and 10 they were treated with HBSS or 100 μg ofanti-CD20-IgG3-mIFNα. Tumor growth was monitored over time.

FIG. 26. Groups of eight mice were injected with 5000 38C13-CD20 cellson day 0. On days 8, 9 and 10 they were treated with HBSS or 100 μg ofanti-CD20-IgG3-mIFNα. Survival was monitored over time.

FIG. 27 shows that repeat dosing enhanced the efficacy ofanti-CD20-mIFNα. Moreover, the data show the surprising result thatattachment of a targeting moiety (e.g., anti-CD20) increased theefficacy of the fusion protein. Mice (n=8 per group) were treated with10 μg of anti-CD-20-mIFNα 5, 6 and 7 days post tumor inoculation. Onegroup of 8 was given additional doses of 30 μg of fusion protein 12 and19 days post tumor inoculation (repeat doses). Mice were followed forsurvival and sacrificed when tumors reached 1.4 cm in diameter as perinstitutional guidelines. Mice treated with HBSS were used as controls.

FIG. 28 shows the effect of the fusion protein on cell proliferation.The fusion protein has interferon activity as measured by the inhibitionof cell proliferation which is improved by targeting. Cells wereincubated with the indicated proteins for 48 hours. Cell proliferationwas measured using the MTS assay and % growth inhibition calculated as[1−(ODexp/ODuntreated0]*100.

FIG. 29, panels A-C, show that anti-tumor efficacy of anti-CD20 requiresIFNAR expression. Panel A: Flow cytometry analysis of IFNAR expression.38C13-CD20 transduced with IFNAR-specific shRNA (38C13-huCD201FNAR KD),38C13 transduced with nonspecific shRNA (38C13-huCD20 control) and38C13-CD20 parental cells stained with anti-IFNAR-biotin primaryantibody (clone MAR1-5Ac) and detected with streptavidin-PE.Biotinylated IgG1 isotype stained control is also shown. Panel B:Apoptosis assay using parental 38C13-huCD20 and 38C13-huCD20IFNAR KD.Cell lines were treated with 1000 pM of anti-CD20-mIFNα and stained withAnnexin V/PI 48 hours later. Panel C: Tumor challenge with38C13-huCD201FNAR KD cells. Mice (n=8) were treated 5, 6 and 7 daysafter tumor inoculation with 10 μg of anti-CD20 mIFNα or the molarequivalent of the indicated proteins. Mice were followed for survivaland sacrificed when tumors reached 1.4 cm in diameter as perinstitutional guidelines. Mice treated with HBSS were used as control.

FIG. 30 shows that anti-CD20-hIFNα has proapoptotic activity against thehuman B-cell lymphoma Daudi. Cells were incubated with varyingconcentrations of the indicated proteins for 72 hours. Staining withAnnexinV-FITC and PI was performed to distinguish necrotic(Annexin⁻PI⁺), early apoptotic (Annexin⁺PI⁻) and late apoptotic(Annexin⁺PI⁺) cell populations. The percentage of total apoptotic cellswas quantified for each sample as the sum of early apoptotic and lateapoptotic cells. Experiments were performed in triplicate and error barsindicate mean±SD. *p=0.0014. **p=0.003.

FIG. 31 shows that anti-CD20-IgG3-huIFNα is effective against rituximabresistant human cell lines. RR1 (Ramos rituximab-resistant) and Ramoscells were treated with 1000 pM of anti-CD20-IgG3-huIFNα for 72 hours.They were then stained with Annexin V-Alexa488 and PI (propidium iodide)to determine % apoptotic cells.

FIG. 32 shows the activity of IFN-α and IFN-β fusion proteins.38C13-CD20 cells were incubated with the various treatments at 37° C. ina 5% CO₂ atmosphere for 72 hours. Cell viability was quantified usingthe MTS assay (Promega) by measuring absorbance at 490 nm using aSynergy HT Multi-Detection Microplate Reader. Data were analyzed bynon-linear regression analysis using Prism GraphPad.

FIG. 33 shows that anti-CD20-hIFNβ is effective against human cells.Daudi cells were incubated with the various treatment at 37° C. in a 5%CO₂ atmosphere for 96 hours. Cell viability was quantified using the MTSassay (Promega) by measuring absorbance at 490 nm using a Synergy HTMulti-Detection Microplate Reader. Data were analyzed by non-linearregression analysis using Prism GraphPad.

FIG. 34 shows that anti-CD20-mIFNβ is effective against cells expressinglow levels of the IFN receptor. 38C13-CD20 cells or 38C13-CD20 cells inwhich the expression of the IFN receptor had been decreased (Knock Down)using shRNA were incubated with the indicated proteins at variousconcentrations for 48 hours. Cell viability was then quantified usingthe MTS assay. Data were analyzed by non-linear regression using PrismGraphPad.

FIG. 35, panels A-D, show that anti-CD20-hIFNα completely curesestablished human xenograft tumors. Panels A-C: Tumor growth in mice(n=5-7 per group) inoculated subcutaneously with Daudi cells and treatedas indicated with three weekly doses of 30 μg fusion protein, theequivalent molar concentration of rituximab, or HBSS. Treatment wasadministered 30, 37 and 44 days post tumor inoculation (arrows) to micewith tumors at least 0.5 cm in diameter. HBSS was injected as a control.Symbols represent individual mice. Panel D: Survival curves for the micewhose tumor growth is shown in panels A-C. *P=0.02.

DETAILED DESCRIPTION

Interferon is an important cytokine in initiating the innate immuneresponse and also demonstrates a wide spectrum of anti-tumor activities.The clinical use of interferon (e.g., IFN-α) as an anticancer drug,however, is hampered by its short half-life, which significantlycompromises its therapeutic effect. In certain embodiments thisinvention pertains to the discovery that the therapeutic index andactual activity (even in vitro) of interferon can be improved byattaching the interferon to a targeting moiety thatspecifically/preferentially binds a marker on or associated with thetarget cell (e.g., a tumor cell). This permits the deliver of higherdoses of interferon to the target site with fewer systemic complicationsand the greater innate activity of the construct provides a greatertherapeutic window. This was illustrated, in certain, by theconstruction and use of a fusion protein consisting of an anti-HER2/neuIgG3 and IFN-α or IFN-β (e.g., anti-HER2/neu-IgG3-IFN-α) and in anotherembodiment by the construction and use of anti-CD20-IFN-α andanti-CD20-IFN-β fusion proteins.

The efficacy of the HER2/neu-IgG3-IFN constructs was tested on a murineB-cell lymphoma, 38C13, transduced with human HER2/neu. Theanti-HER2/neu-IgG3-IFN fusion protein exhibited a potent effect ininhibiting the 38C13/HER2 tumor growth in vivo, and even administrationof 1 μg anti-HER2/neu IgG3-IFN-α resulted in 88% of long-term survivorsafter tumor challenge.

Remarkably, anti-HER2/neu IgG3-IFN-α demonstrated a potent activityagainst established 38C13/HER2 tumors, and complete tumor remission wasobserved in 88% treated mice. This dramatic anti-tumor activity wasmediated by IFN-α induced apoptosis and targeting IFN-α to 38C13/HER2tumor cells by the anti-HER2/neu IgG3 antibody was essential topotentiate these effects.

Similar results were observed for the anti-CD20-IgG3-IFN-α constructsand anti-CD20-IFN-β constructs (see, Examples herein). These resultsindicate that attachment (e.g., fusion) of an interferon (e.g., IFN-α)to a targeting moiety (e.g., to a tumor specific antibody) produces aneffective therapeutic that can be used to inhibit the growth and/orproliferation or even to kill target cell(s). Thus, for example, theexemplary constructs described herein can readily be used for treatmentof B cell lymphoma and other cancers in clinic.

Thus, in certain embodiments, this invention provides constructs (e.g.chimeric moieties) comprising an interferon (e.g., IFN-α, IFN-β, etc.)attached to a targeting moiety (e.g., to an antibody that specificallybinds a cancer specific marker on a cancer cell). The constructs includechemical conjugates as well as fusion proteins. Also provided arenucleic acids encoding the fusion proteins as well as cells transfectedwith the nucleic acids to express the fusion proteins. Also provided aremethods of inhibiting growth and proliferation of cancer cells as wellas kits comprising, e.g. the chimeric moieties described herein, for thetreatment of various cancers.

I. Chimeric Constructs Comprising a Targeting Moiety Attached to anInterferon.

It was a surprising discovery that chimeric constructs comprising atargeting moiety (e.g., an anti-tumor marker antibody) attached to anative (wild type) or modified IFN (e.g., IFN-α, IFN-β, etc.) can beeffectively used to inhibit the growth and/or proliferation of targetcancer cells expressing or associated with the marker to which thetargeting moiety is directed. In certain embodiments the targetingmoieties are chemically conjugated to the interferon, while in otherembodiments, the targeting moiety is expressed as a fusion protein withthe interferon. When produced as a fusion protein the targeting moiety(e.g., antibody) component can be directly fused to the IFN-α orattached by means of a peptide linker (e.g., a (Gly₄Ser)₃ (SEQ ID NO:5)linker, a Gly₄Ser (SEQ ID NO:6) linker, an AEAAAKEAAAKA (SEQ ID NO:7)linker, and the like.

Illustrative nucleic acid and amino acid constructs used in thecompositions and methods described herein are shown in FIG. 1 and inTable 1. It will be appreciated that while the constructs in this figureare shown with particular linkers, targeting moieties and interferons,in certain embodiments other linkers, other targeting moieties and otherinterferons can be substituted therefore as described herein.

TABLE 1 Various illustrative constructs used in certain embodimentsdescribed herein. SEQ ID Description and Sequence NO:αCD20 light chain - nucleic acid sequence:ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCA 8GTCAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAAαCD20 light chain - amino acid sequence:MKLPVRLLVLMFWIPASSSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQ 9QKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWT SNPPTFGGGTKLEIKαCD20-IgG3-muIFNα Gly₄Ser- nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 10GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGACCTGCCTCAGACTCATAACCTCAGGAACAAGAGAGCCTTGACACTCCTGGTACAAATGAGGAGACTCTCCCCTCTCTCCTGCCTGAAGGACAGGAAGGACTTTGGATTCCCGCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCTCAAGCCATCCCTGTCCTGAGTGAGCTGACCCAGCAGATCCTGAACATCTTCACATCAAAGGACTCATCTGCTGCTTGGAATGCAACCCTCCTAGACTCATTCTGCAATGACCTCCACCAGCAGCTCAATGACCTGCAAGGTTGTCTGATGCAGCAGGTGGGGGTGCAGGAATTTCCCCTGACCCAGGAAGATGCCCTGCTGGCTGTGAGGAAATACTTCCACAGGATCACTGTGTACCTGAGAGAGAAGAAACACAGCCCCTGTGCCTGGGAGGTGGTCAGAGCAGAAGTCTGGAGAGCCCTGTCTTCCTCTGCCAATGTGCTGGGAAGACTGAGAGAAGAGAAA αCD20-IgG3-muIFNαGly₄Ser linker - Amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 11VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGKS GGGGS CDLPQTHNLRNKRALTLLVQMRRLSPLSCLKDRKDFGFPQEKVDAQQIKKAQAIPVLSELTQQILNIFTSKDSSAAWNATLLDSFCNDLHQQLNDLQGCLMQQVGVQEFPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSSANVLGRLREEK αCD20-IgG3-muIFNαalpha helical linker - nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 12GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGACCTGCCTCAGACTCATAACCTCAGGAACAAGAGAGCCTTGACACTCCTGGTACAAATGAGGAGACTCTCCCCTCTCTCCTGCCTGAAGGACAGGAAGGACTTTGGATTCCCGCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCTCAAGCCATCCCTGTCCTGAGTGAGCTGACCCAGCAGATCCTGAACATCTTCACATCAAAGGACTCATCTGCTGCTTGGAATGCAACCCTCCTAGACTCATTCTGCAATGACCTCCACCAGCAGCTCAATGACCTGCAAGGTTGTCTGATGCAGCAGGTGGGGGTGCAGGAATTTCCCCTGACCCAGGAAGATGCCCTGCTGGCTGTGAGGAAATACTTCCACAGGATCACTGTGTACCTGAGAGAGAAGAAACACAGCCCCTGTGCCTGGGAGGTGGTCAGAGCAGAAGTCTGGAGAGCCCTGTCTTCCTCTGCCAATGTGCTGGGAAGACTGAGAGAAGAGAA AαCD20-IgG3-muIFNα alpha helical linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 13VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGKAEAAAKEAAAKAGSCDLPQTHNLRNKRALTLLVQMRRLSPLSCLKDRKDFGFPQEKVDAQQIKKAQAIPVLSELTQQILNIFTSKDSSAAWNATLLDSFCNDLHQQLNDLQGCLMQQVGVQEFPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSSANVLGRLREEK αCD20-IgG3-huIFNαGly₄Ser linker - nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 14GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA αCD20-IgG3-huIFNαGly₄Ser linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 15VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE αCD20-IgG3-huIFNαalpha helical linker - nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 16GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA αCD20-IgG3-huIFNαalpha helical linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 17VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGK AEAAAKEAAAKA GSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE αCD20-IgG1-muIFNαGly₄Ser linker - nucleic acid sequenceATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 18GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGACCTGCCTCAGACTCATAACCTCAGGAACAAGAGAGCCTTGACACTCCTGGTACAAATGAGGAGACTCTCCCCTCTCTCCTGCCTGAAGGACAGGAAGGACTTTGGATTCCCGCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCTCAAGCCATCCCTGTCCTGAGTGAGCTGACCCAGCAGATCCTGAACATCTTCACATCAAAGGACTCATCTGCTGCTTGGAATGCAACCCTCCTAGACTCATTCTGCAATGACCTCCACCAGCAGCTCAATGACCTGCAAGGTTGTCTGATGCAGCAGGTGGGGGTGCAGGAATTTCCCCTGACCCAGGAAGATGCCCTGCTGGCTGTGAGGAAATACTTCCACAGGATCACTGTGTACCTGAGAGAGAAGAAACACAGCCCCTGTGCCTGGGAGGTGGTCAGAGCAGAAGTCTGGAGAGCCCTGTCTTCCTCTGCCAATGTGCTGGGAAGACTGAGAGAAGAGAA AαCD20-IgG1-muIFNα Gly₄Ser linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 19VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKS GGGGS CDLPQTHNLRNKRALTLLVQMRRLSPLSCLKDRKDFGFPQEKVDAQQIKKAQAIPVLSELTQQILNIFTSKDSSAAWNATLLDSFCNDLHQQLNDLQGCLMQQVGVQEFPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSSANVLGRLREEK αCD20-IgG1-muIFNαalpha helical linker - nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 20GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGACCTGCCTCAGACTCATAACCTCAGGAACAAGAGAGCCTTGACACTCCTGGTACAAATGAGGAGACTCTCCCCTCTCTCCTGCCTGAAGGACAGGAAGGACTTTGGATTCCCGCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCTCAAGCCATCCCTGTCCTGAGTGAGCTGACCCAGCAGATCCTGAACATCTTCACATCAAAGGACTCATCTGCTGCTTGGAATGCAACCCTCCTAGACTCATTCTGCAATGACCTCCACCAGCAGCTCAATGACCTGCAAGGTTGTCTGATGCAGCAGGTGGGGGTGCAGGAATTTCCCCTGACCCAGGAAGATGCCCTGCTGGCTGTGAGGAAATACTTCCACAGGATCACTGTGTACCTGAGAGAGAAGAAACACAGCCCCTGTGCCTGGGAGGTGGTCAGAGCAGAAGTCTGGAGAGCCCTGTCTTCCTCTGCCAATGTGCTGGGAAGACTGAGAGAAGAGAAATGA αCD20-IgG1-muIFNαalpha helical linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 21VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AEAAAKEAAAKA GSCDLPQTHNLRNKRALTLLVQMRRLSPLSCLKDRKDFGFPQEKVDAQQIKKAQAIPVLSELTQQILNIFTSKDSSAAWNATLLDSFCNDLHQQLNDLQGCLMQQVGVQEFPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSSANVLGRLREEK αCD20-IgG1-huIFNαGly₄Ser linker - nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 22GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA αCD20-IgG1-huIFNαGly₄Ser linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 23VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKS GGGGS CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE αCD20-IgG1-huIFNαalpha helical linker - nucleic acid sequence:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 24GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAATGA αCD20-IgG1-huIFNαalpha helical linker - amino acid sequence:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 25VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AEAAAKEAAAKA GSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEαHer2/neu light chain - nucleic acid sequence:ATGGGATGGAGCTGGGTAATCCTCTTTCTCCTGTCAGTAACTGCAGGTGTCCACT 26CCCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTATGATCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCCTCCTGGGACTACACCCTCTCGGGCTGGGTGTTCGGAGGAGGGACCAAGGTCACCGTCCTAGGTCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGαHer2/neu light chain - Amino acid sequenceMGWSWVILFLLSVTAGVHSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSW 27YQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKVTVLGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC αHer2/neu-IgG1-muIFNαGly₄Ser linker - Nucleic acid sequence:ATGGGATGGAGCTGGGTAATGCATCTTTCTCCTGTCAGTAACTGCGGTGTCCACT 28CCCAGGTCCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCACCGACCGGACTTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGACCTGCCTCAGACTCATAACCTCAGGAACAAGAGAGCCTTGACACTCCTGGTACAAATGAGGAGACTCTCCCCTCTCTCCTGCCTGAAGGACAGGAAGGACTTTGGATTCCCGCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCTCAAGCCATCCCTGTCCTGAGTGAGCTGACCCAGCAGATCCTGAACATCTTCACATCAAAGGACTCATCTGCTGCTTGGAATGCAACCCTCCTAGACTCATTCTGCAATGACCTCCACCAGCAGCTCAATGACCTGCAAGGTTGTCTGATGCAGCAGGTGGGGGTGCAGGAATTTCCCCTGACCCAGGAAGATGCCCTGCTGGCTGTGAGGAAATACTTCCACAGGATCACTGTGTACCTGAGAGAGAAGAAACACAGCCCCTGTGCCTGGGAGGTGGTCAGAGCAGAAGTCTGGAGAGCCCTGTCTTCCTCTGCCAATGTGCTGGGAAGACTGAGAGAAGAGAAA αHer2/neu-IgG1mIFNαGly₄Ser linker amino acid sequence:MGWSWVMHLSPVSNCGVHSQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAW 29VRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSCDLPQTHNLRNKRALTLLVQMRRLSPLSCLKDRKDFGFPQEKVDAQQIKKAQAIPVLSELTQQILNIFTSKDSSAAWNATLLDSFCNDLHQQLNDLQGCLMQQVGVQEFPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSSANVLGRLREEKαHer2/neu-IgG1-muIFNa alpha helix linker - Nucleic acid sequence:ATGGGATGGAGCTGGGTAATGCATCTTTCTCCTGTCAGTAACTGCGGTGTCCACT 30CCCAGGTCCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCACCGACCGGACTTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGACCTGCCTCAGACTCATAACCTCAGGAACAAGAGAGCCTTGACACTCCTGGTACAAATGAGGAGACTCTCCCCTCTCTCCTGCCTGAAGGACAGGAAGGACTTTGGATTCCCGCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCTCAAGCCATCCCTGTCCTGAGTGAGCTGACCCAGCAGATCCTGAACATCTTCACATCAAAGGACTCATCTGCTGCTTGGAATGCAACCCTCCTAGACTCATTCTGCAATGACCTCCACCAGCAGCTCAATGACCTGCAAGGTTGTCTGATGCAGCAGGTGGGGGTGCAGGAATTTCCCCTGACCCAGGAAGATGCCCTGCTGGCTGTGAGGAAATACTTCCACAGGATCACTGTGTACCTGAGAGAGAAGAAACACAGCCCCTGTGCCTGGGAGGTGGTCAGAGCAGAAGTCTGGAGAGCCCTGTCTTCCTCTGCCAATGTGCTGGGAAGACTGAGAGAAGAGAAAαHer2/neu-IgG1mIFNa alpha helix linker amino acid sequence:MGWSWVMHLSPVSNCGVHSQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAW 31VRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAEAAAKEAAAKAGSCDLPQTHNLRNKRALTLLVQMRRLSPLSCLKDRKDFGFPQEKVDAQQIKKAQAIPVLSELTQQILNIFTSKDSSAAWNATLLDSFCNDLHQQLNDLQGCLMQQVGVQEFPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSSANVLGRLREEKαHer2/neu-IgG2hIFNα Gly₄Ser linker nuclei acid sequence:ATGGGATGGAGCTGGGTAATGCATCTTTCTCCTGTCAGTAACTGCGGTGTCCACT 32CCCAGGTCCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCACCGACCGGACTTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA αHer2/neu-IgG2hIFNαGly₄Ser linker amino acid sequenceMGWSWVMHLSPVSNCGVHSQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAW 33VRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE αHer2/neu-IgG1-huIFNαalpha helix linker - nucleic acid sequence:ATGGGATGGAGCTGGGTAATGCATCTTTCTCCTGTCAGTAACTGCGGTGTCCACT 34CCCAGGTCCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCACCGACCGGACTTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAATGA αHer2/neu-Ig1hIFNαalpha helix linker amino acid sequence:MGWSWVMHLSPVSNCGVHSQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAW 35VRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAEAAAKEAAAKAGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKENucleotide sequence of anti-CD20 IgG1 GS1 human IFN beta:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 36GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAA CTGAAmino acid sequence of anti-CD20 IgG1 GS1 human IFN beta:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 37VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNNucleotide sequence of anti-CD20 IgG3 GS1 human IFN beta:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 38GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAACTGAAmino acid sequence of anti-CD20 IgG3 GS1 human IFN beta:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 39VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNNucleotide sequence of anti-CD20 IgG3 GS1 murine IFN beta:ATGTACTTGGGACTGAACTGTGTAATCATAGTTTTTCTCTTAAAAGGTGTCCAGA 40GTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACACCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGCACCTGAACTCCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCATCAACTATAAGCAGCTCCAGCTCCAAGAAAGGACGAACATTCGGAAATGTCAGGAGCTCCTGGAGCAGCTGAATGGAAAGATCAACCTCACCTACAGGGCGGACTTTAAGATCCCTATGGAGATGACGGAGAAGATGCAGAAGAGTTACACTGCCTTTGCCATCCAAGAGATGCTCCAGAATGTCTTTCTTGTCTTCAGAAACAATTTCTCCAGCACTGGGTGGAATGAGACTATTGTTGTACGTCTCCTGGATGAACTCCACCAGCAGACAGTGTTTCTGAAGACAGTACTAGAGGAAAAGCAAGAGGAAAGATTGACGTGGGAGATGTCCTCAACTGCTCTCCACTTGAAGAGCTATTACTGGAGGGTGCAAAGGTACCTTAAACTCATGAAGTACAACAGCTACGCCTGGATGGTGGTCCGAGCAGAGATCTTCAGGAACTTTCTCATCATTCGAAGACTTACCAGAAACTTCCAAAACTGAAmino acid sequence of anti-CD20 IgG3 GS1 murine IFN beta:MYLGLNCVIIVFLLKGVQSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHW 41VKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSINYKQLQLQERTNIRKCQELLEQLNGKINLTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETIVVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLKLMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNAnti-HER2/neu IgG1 G/S hIFN alpha - nucleotide sequence:ATGGAATGCAGCTGGGTAATGCTCTTTCTCCTGTCAGTAACTGCAGGTGTCCACT 42CCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAATGAAnti-HER2/neu IgG1 G/S huIFN alpha amino acid sequence:MECSWVMLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW 43VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKS GGGGS CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAnti-HER/neu IgG1 G/S huIFN Beta nucleotide sequence:ATGGAATGCAGCTGGGTAATGCTCTTTCTCCTGTCAGTAACTGCAGGTGTCCACT 44CCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCTGGTGGCGGTGGATCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAACTG AAnti-HER/neu IgG1 G/S hIFN Beta amino acid sequence:MECSWVMLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW 45VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKS GGGGS MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN Anti-her2/neu IgG1 alpha helical huIFN alpha nucleic acid sequence:ATGGAATGCAGCTGGGTAATGCTCTTTCTCCTGTCAGTAACTGCAGGTGTCCACT 46CCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGCAGAGGCCGCAGCTAAAGAGGCCGCAGCCAAAGCGGGATCCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAATGAAnti-her2/neu IgG1 alpha helical huIFN alpha amino acid sequence:MECSWVMLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW 47VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AEAAAKEAAAKA GSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEAnti-HER2/neu Light chain - nucleic acid sequence:ATGGAATGGAGCTGTGTCATGCTCTTTCTCCTGTCAGTAACTGCAGGTGTCCACT 48CCGACATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGATCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAnti-HER2/neu Light chain - amino acid sequence:MEWSCVMLFLLSVTAGVHSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY 49QQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECWhile the constructs in this Table are shown with particular targetingmoieties, particular linkers, and particular interferons, in certainembodiments other targeting moieties, linkers, and interferons can besubstituted therefore as described herein.

A) Targeting Moieties.

In various embodiments, the targeting moiety is a molecule thatspecifically or preferentially binds a marker expressed by (e.g., on thesurface of) or associated with the target cell(s). While essentially anycell can be targeted, certain preferred cells include those associatedwith a pathology characterized by hyperproliferation of a cell (i.e., ahyperproliferative disorder). Illustrative hyperproliferative disordersinclude, but are not limited to psoriasis, neutrophilia, polycythemia,thrombocytosis, and cancer.

Hyperproliferative disorders characterized as cancer include but are notlimited to solid tumors, such as cancers of the breast, respiratorytract, brain, reproductive organs, digestive tract, urinary tract, eye,liver, skin, head and neck, thyroid, parathyroid and their distantmetastases. These disorders also include lymphomas, sarcomas, andleukemias. Examples of breast cancer include, but are not limited toinvasive ductal carcinoma, invasive lobular carcinoma, ductal carcinomain situ, and lobular carcinoma in situ. Examples of cancers of therespiratory tract include, but are not limited to small-cell andnon-small-cell lung carcinoma, as well as bronchial adenoma andpleuropulmonary blastoma. Examples of brain cancers include, but are notlimited to brain stem and hypophtalmic glioma, cerebellar and cerebralastrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal andpineal tumor. Tumors of the male reproductive organs include, but arenot limited to prostate and testicular cancer. Tumors of the femalereproductive organs include, but are not limited to endometrial,cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of theuterus. Tumors of the digestive tract include, but are not limited toanal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic,rectal, small-intestine, and salivary gland cancers. Tumors of theurinary tract include, but are not limited to bladder, penile, kidney,renal pelvis, ureter, and urethral cancers. Eye cancers include, but arenot limited to intraocular melanoma and retinoblastoma. Examples ofliver cancers include, but are not limited to hepatocellular carcinoma(liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma. Skin cancers include, but are notlimited to squamous cell carcinoma, Kaposi's sarcoma, malignantmelanoma, Merkel cell skin cancer, and non-melanoma skin cancer.Head-and-neck cancers include, but are not limited tolaryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and lipand oral cavity cancer. Lymphomas include, but are not limited toAIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-celllymphoma, Hodgkin's disease, and lymphoma of the central nervous system.Sarcomas include, but are not limited to sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma. Leukemias include, but are not limited to acutemyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, and hairy cell leukemia.

These disorders have been well characterized in humans, but also existwith a similar etiology in other mammals, and can be treated byadministering pharmaceutical compositions of the present invention.

In certain embodiments, the targeting moiety is a moiety that binds acancer marker (e.g., a tumor associated antigen). A wide variety ofcancer markers are known to those of skill in the art. The markers neednot be unique to cancer cells, but can also be effective where theexpression of the marker is elevated in a cancer cell (as compared tonormal healthy cells) or where the marker is not present at comparablelevels in surrounding tissues (especially where the chimeric moiety isdelivered locally).

Illustrative cancer markers include, for example, the tumor markerrecognized by the ND4 monoclonal antibody. This marker is found onpoorly differentiated colorectal cancer, as well as gastrointestinalneuroendocrine tumors (see, e.g., Tobi et al. (1998) Cancer Detectionand Prevention, 22(2): 147-152). Other important targets for cancerimmunotherapy are membrane bound complement regulatory glycoprotein:CD46, CD55 and CD59, which have been found to be expressed on most tumorcells in vivo and in vitro. Human mucins (e.g. MUC1) are known tumormarkers as are gp100, tyrosinase, and MAGE, which are found in melanoma.Wild-type Wilms' tumor gene WT1 is expressed at high levels not only inmost of acute myelocytic, acute lymphocytic, and chronic myelocyticleukemia, but also in various types of solid tumors including lungcancer.

Acute lymphocytic leukemia has been characterized by the TAAs HLA-Dr,CD1, CD2, CD5, CD7, CD19, and CD20. Acute myelogenous leukemia has beencharacterized by the TAAs HLA-Dr, CD7, CD13, CD14, CD15, CD33, and CD34.Breast cancer has been characterized by the markers EGFR, HER2, MUC1,Tag-72. Various carcinomas have been characterized by the markers MUC1,TAG-72, and CEA. Chronic lymphocytic leukemia has been characterized bythe markers CD3, CD19, CD20, CD21, CD25, and HLA-DR. Hairy cell leukemiahas been characterized by the markers CD19, CD20, CD21, CD25. Hodgkin'sdisease has been characterized by the Leu-M1 marker. Various melanomashave been characterized by the HMB 45 marker. Non-hodgkins lymphomashave been characterized by the CD20, CD19, and Ia marker. And variousprostate cancers have been characterized by the PSMA and SE10 markers.

In addition, many kinds of tumor cells display unusual antigens that areeither inappropriate for the cell type and/or its environment, or areonly normally present during the organisms' development (e.g. fetalantigens). Examples of such antigens include the glycosphingolipid GD2,a disialoganglioside that is normally only expressed at a significantlevel on the outer surface membranes of neuronal cells, where itsexposure to the immune system is limited by the blood-brain barrier. GD2is expressed on the surfaces of a wide range of tumor cells includingneuroblastoma, medulloblastomas, astrocytomas, melanomas, small-celllung cancer, osteosarcomas and other soft tissue sarcomas. GD2 is thus aconvenient tumor-specific target for immunotherapies.

Other kinds of tumor cells display cell surface receptors that are rareor absent on the surfaces of healthy cells, and which are responsiblefor activating cellular signaling pathways that cause the unregulatedgrowth and division of the tumor cell. Examples include (ErbB2)HER2/neu, a constitutively active cell surface receptor that is producedat abnormally high levels on the surface of breast cancer tumor cells.

Other useful targets include, but are not limited to CD20, CD52, CD33,epidermal growth factor receptor and the like.

An illustrative, but not limiting list of suitable tumor markers isprovided in Table 2. Antibodies to these and other cancer markers areknown to those of skill in the art and can be obtained commercially orreadily produced, e.g. using phage-display technology.

TABLE 2 Illustrative cancer markers and associated references, all ofwhich are incorporated herein by reference for the purpose ofidentifying the referenced tumor markers. Marker Reference 5 alphareductase Délos et al. (1998) Int J Cancer, 75: 6 840-846 α-fetoproteinEsteban et al. (1996) Tumour Biol., 17(5): 299-305 AM-1 Harada et al.(1996) Tohoku J Exp Med., 180(3): 273-288 APC Dihlmann et al. (1997)Oncol Res., 9(3) 119-127 APRIL Sordat et al. ({grave over ( )}998) J ExpMed., 188(6): 1185-1190 BAGE Böel et al. (1995) Immunity, 2: 167-175.β-catenin Hugh et al. (1999) Int J Cancer, 82(4): 504-11 Bc12 Koty etal. (1999) Lung Cancer, 23(2): 115-127 bcr-abl (b3a2) Verfaillie et al.({grave over ( )}996) Blood, 87(11): 4770-4779 CA-125 Bast et al.({grave over ( )}998) Int J Biol Markers, 13(4): 179-187 CASP-8/FLICEMandruzzato et al. (1997) J Exp Med., 186(5): 785-793. CathepsinsThomssen et al. (1995) Clin Cancer Res., 1(7): 741-746 CD19 Scheuermannet al. (1995) Leuk Lymphoma, 18(5-6): 385-397 CD20 Knox et al. (1996)Clin Cancer Res., 2(3): 457-470 CD21, CD23 Shubinsky et al. (1997) LeukLymphoma, 25(5-6): 521-530 CD22, CD38 French et al. (1995) Br J Cancer,71(5): 986-994 CD33 Nakase et al. (1996) Am J Clin Pathol., 105(6):761-768 CD35 Yamakawa et al. Cancer, 73(11): 2808-2817 CD44 Naot et al.(1997) Adv Cancer Res., 71: 241-319 CD45 Buzzi et al. (1992) CancerRes., 52(14): 4027-4035 CD46 Yamakawa et al. (1994) Cancer, 73(11):2808-2817 CD5 Stein et al. (1991) Clin Exp Immunol., 85(3): 418-423 CD52Ginaldi et al. (1998) Leuk Res., 22(2): 185-191 CD55 Spendlove et al.(1999) Cancer Res., 59: 2282-2286. CD59 (791Tgp72) Jarvis et al. (1997)Int J Cancer, 71(6): 1049-1055 CDC27 Wang et al. (1999) Science,284(5418): 1351-1354 CDK4 Wölfel et al. (1995) Science, 269(5228):1281-1284 CEA Kass et al. (1999) Cancer Res., 59(3): 676-683 c-mycWatson et al. (1991) Cancer Res., 51(15): 3996-4000 Cox-2 Tsujii et al.(1998) Cell, 93: 705-716 DCC Gotley et al. (1996) Oncogene, 13(4):787-795 DcR3 Pitti et al. (1998) Nature, 396: 699-703 E6/E7 Steller etal. (1996) Cancer Res., 56(21): 5087-5091 EGFR Yang et al. (1999) CancerRes., 59(6): 1236-1243. EMBP Shiina et al. (1996) Prostate, 29(3):169-176. Ena78 Arenberg et al. (1998) J. Clin. Invest., 102: 465-472.FGF8b and FGF8a Dorkin et al. (1999) Oncogene, 18(17): 2755-2761FLK-1/KDR Annie and Fong (1999) Cancer Res., 59: 99-106 Folic AcidReceptor Dixon et al. (1992) J Biol Chem., 267(33): 24140-72414 G250Divgi et al. (1998) Clin Cancer Res., 4(11): 2729-2739 GAGE-Family DeBacker et al. (1999) Cancer Res., 59(13): 3157-3165 gastrin 17 Watson etal. (1995) Int J Cancer, 61(2): 233-240 Gastrin-releasing Wang et al.(1996) Int J Cancer, 68(4): 528-534 hormone (bombesin) GD2/GD3/GM2Wiesner and Sweeley (1995) Int J Cancer, 60(3): 294-299 GnRH Bahk et al.(1998) Urol Res., 26(4): 259-264 GnTV Hengstler et al. (1998) RecentResults Cancer Res., 154: 47-85 gp100/Pmel17 Wagner et al. (1997) CancerImmunol Immunother., 44(4): 239-247 gp-100-in4 Kirkin et al. (1998)APMIS, 106(7): 665-679 gp15 Maeurer et al.(1996) Melanoma Res., 6(1):11-24 gp75/TRP-1 Lewis et al. (1995) Semin Cancer Biol., 6(6): 321-327hCG Hoermann et al. (1992) Cancer Res., 52(6): 1520-1524 HeparanaseVlodavsky et al. (1999) Nat Med., 5(7): 793-802 Her2/neu Lewis et al.(1995) Semin Cancer Biol., 6(6): 321-327 Her3 HMTV Kahl et al. (1991) BrJ Cancer, 63(4): 534-540 Hsp70 Jaattela et al. (1998) EMBO J., 17(21):6124-6134 hTERT Vonderheide et al. (1999) Immunity, 10: 673-679. 1999.(telomerase) IGFR1 Ellis et al. (1998) Breast Cancer Res. Treat., 52:175-184 IL-13R Murata et al. (1997) Biochem Biophys Res Commun., 238(1):90-94 iNOS Klotz et al. (1998) Cancer, 82(10): 1897-1903 Ki 67 Gerdes etal. (1983) Int J Cancer, 31: 13-20 KIAA0205 Guéguen et al. (1998) JImmunol., 160(12): 6188-6194 K-ras, H-ras, Abrams et al. (1996) SeminOncol., 23(1): 118-134 N-ras KSA Zhang et al. (1998) Clin Cancer Res.,4(2): 295-302 (CO17-1A) LDLR-FUT Caruso et al. (1998) Oncol Rep., 5(4):927-930 MAGE Family Marchand et al. (1999) Int J Cancer, 80(2): 219-230(MAGE1, MAGE3, etc.) Mammaglobin Watson et al. (1999) Cancer Res., 59:13 3028-3031 MAP17 Kocher et al. (1996) Am J Pathol., 149(2): 493-500Melan-A/ Lewis and Houghton (1995) Semin Cancer Biol., 6(6): 321-327MART-1 mesothelin Chang et al. (1996) Proc. Natl. Acad. Sci., USA,93(1): 136-140 MIC A/B Groh et al. (1998) Science, 279: 1737-1740MT-MMP's, such as Sato and Seiki (1996) J Biochem (Tokyo), 119(2):209-215 MMP2, MMP3, MMP7, MMP9 Mox1 Candia et al. (1992) Development,116(4): 1123-1136 Mucin, such as MUC- Lewis and Houghton (1995) SeminCancer Biol., 6(6): 321-327 1, MUC-2, MUC-3, and MUC-4 MUM-1 Kirkin etal. (1998) APMIS, 106(7): 665-679 NY-ESO-1 Jager et al. (1998) J. Exp.Med., 187: 265-270 Osteonectin Graham et al. (1997) Eur J Cancer,33(10): 1654-1660 p15 Yoshida et al. (1995) Cancer Res., 55(13):2756-2760 P170/MDR1 Trock et al. (1997) J Natl Cancer Inst., 89(13):917-931 p53 Roth et al. (1996) Proc. Natl. Acad. Sci., USA, 93(10):4781-4786. p97/melanotransferrin Furukawa et al. (1989) J Exp Med.,169(2): 585-590 PAI-1 Grøndahl-Hansen et al. (1993) Cancer Res., 53(11):2513-2521 PDGF Vassbotn et al. (1993) Mol Cell Biol., 13(7): 4066-4076Plasminogen (uPA) Naitoh et al. (1995) Jpn J Cancer Res., 86(1): 48-56PRAME Kirkin et al. (1998) APMIS, 106(7): 665-679 Probasin Matuo et al.(1985) Biochem Biophys Res Commun., 130(1): 293-300 Progenipoietin — PSASanda et al. (1999) Urology, 53(2): 260-266. PSM Kawakami et al. (1997)Cancer Res., 57(12): 2321-2324 RAGE-1 Gaugler et al. (1996)Immunogenetics, 44(5): 323-330 Rb Dosaka-Akita et al. (1997) Cancer,79(7): 1329-1337 RCAS1 Sonoda et al. (1996) Cancer, 77(8): 1501-1509.SART-1 Kikuchi et al. (1999) Int J Cancer, 81(3): 459-466 SSX gene Gureet al. (1997) Int J Cancer, 72(6): 965-971 family STAT3 Bromberg et al.(1999) Cell, 98(3): 295-303 STn Sandmaier et al. (1999) J Immunother.,22(1): 54-66 (mucin assoc.) TAG-72 Kuroki et al. (1990) Cancer Res.,50(16): 4872-4879 TGF-α Imanishi et al. (1989) Br J Cancer, 59(5):761-765 TGF-β Picon et al. (1998) Cancer Epidemiol Biomarkers Prey,7(6): 497-504 Thymosin β 15 Bao et al. (1996) Nature Medicine. 2(12),1322-1328 IFN-α Moradi et al. (1993) Cancer, 72(8): 2433-2440 TPAMaulard et al. (1994) Cancer, 73(2): 394-398 TPI Nishida et al. (1984)Cancer Res 44(8): 3324-9 TRP-2 Parkhurst et al. (1998) Cancer Res.,58(21) 4895-4901 Tyrosinase Kirkin et al. (1998) APMIS, 106(7): 665-679VEGF Hyodo et al. (1998) Eur J Cancer, 34(13): 2041-2045 ZAG Sanchez etal. (1999) Science, 283(5409): 1914-1919 p16INK4 Quelle et al. (1995)Oncogene Aug. 17, 1995; 11(4): 635-645 Glutathione Hengstler (1998) etal. Recent Results Cancer Res., 154: 47-85 S-transferase

Any of the foregoing markers can be used as targets for the targetingmoieties comprising the interferon-targeting moiety constructs of thisinvention. In certain embodiments the target markers include, but arenot limited to members of the epidermal growth factor family (e.g.,HER2, HER3, EGF, HER4), CD1, CD2, CD3, CD5, CD7, CD13, CD14, CD15, CD19,CD20, CD21, CD23, CD25, CD33, CD34, CD38, 5E10, CEA, HLA-DR, HM 1.24,HMB 45, 1a, Leu-M1, MUC1, PMSA, TAG-72, phosphatidyl serine antigen, andthe like.

The foregoing markers are intended to be illustrative and not limiting.Other tumor associated antigens will be known to those of skill in theart.

Where the tumor marker is a cell surface receptor, ligand to thatreceptor can function as targeting moieties. Similarly mimetics of suchligands can also be used as targeting moieties.

Antibodies.

In certain embodiments, the targeting moieties can comprise antibodies,unibodies, or affybodies that specifically or preferentially bind thetumor marker. Antibodies that specifically or preferentially bind tumormarkers are well known to those of skill in the art. Thus, for example,antibodies that bind the CD22 antigen expressed on human B cells includeHD6, RFB4, UV22-2, To15, 4 KB128, a humanized anti-CD22 antibody (hLL2)(see, e.g., Li et al. (1989) Cell. Immunol. 111: 85-99; Mason et al.(1987) Blood 69: 836-40; Behr et al. (1999) Clin. Cancer Res. 5:3304s-3314s; Bonardi et al. (1993) Cancer Res. 53: 3015-3021).

Antibodies to CD33 include for example, HuM195 (see, e.g., Kossman etal. (1999) Clin. Cancer Res. 5: 2748-2755), CMA-676 (see, e.g., Sieverset al., (1999) Blood 93: 3678-3684.

Antibodies to CD38 include for example, AT13/5 (see, e.g., Ellis et al.(1995) J. Immunol. 155: 925-937), HB7, and the like.

In certain embodiments the targeting moiety comprises an anti-HER2antibody. The erb-b2 gene, more commonly known as (Her-2/neu), is anoncogene encoding a transmembrane receptor. Several antibodies have beendeveloped against Her-2/neu, including trastuzumab (e.g., HERCEPTIN®;Formier et al. (1999) Oncology (Huntingt) 13: 647-58), TAB-250(Rosenblum et al. (1999) Clin. Cancer Res. 5: 865-874), BACH-250 (Id.),TA1 (Maier et al. (1991) Cancer Res. 51: 5361-5369), and the mAbsdescribed in U.S. Pat. Nos. 5,772,997; 5,770,195 (mAb 4D5; ATCC CRL10463); and U.S. Pat. No. 5,677,171

Illustrative anti-MUC-1 antibodies include, but are not limited to Mc5(see, e.g., Peterson et al. (1997) Cancer Res. 57: 1103-1108; Ozzello etal. (1993) Breast Cancer Res. Treat. 25: 265-276), and hCTMO1 (see,e.g., Van H of et al. (1996) Cancer Res. 56: 5179-5185).

Illustrative anti-TAG-72 antibodies include, but are not limited to CC49(see, e.g., Pavlinkova et al. (1999) Clin. Cancer Res. 5: 2613-2619),B72.3 (see, e.g., Divgi et al. (1994) Nucl. Med. Biol. 21: 9-15), andthose disclosed in U.S. Pat. No. 5,976,531.

Illustrative anti-HM1.24 antibodies include, but are not limited to amouse monoclonal anti-HM1.24 IgG_(2a)/κ and a humanized anti-HM1.24IgG₁/κ. antibody (see, e.g., Ono et al. (1999) Mol. Immunol. 36:387-395).

A number of antibodies have been developed that specifically bind HER2and some are in clinical use. These include, for example, trastuzumab(e.g., HERCEPTIN®, Formier et al. (1999) Oncology (Huntingt) 13:647-658), TAB-250 (Rosenblum et al., (1999) Clin. Cancer Res. 5:865-874), BACH-250 (Id.), TA1 (see, e.g., Maier et al. (1991) CancerRes. 51: 5361-5369), and the antibodies described in U.S. Pat. Nos.5,772,997; 5,770,195, and 5,677,171.

Other fully human anti-HER2/neu antibodies are well known to those ofskill in the art. Such antibodies include, but are not limited to the C6antibodies such as C6.5, DPL5, G98A, C6 MH3-B1, B1D2, C6VLB, C6VLD,C6VLE, C6VLF, C6 MH3-D7, C6 MH3-D6, C6 MH3-D5, C6 MH3-D3, C6 MH3-D2, C6MH3-D1, C6 MH3-C4, C6 MH3-C3, C6 MH3-B9, C6 MH3-B5, C6 MH3-B48, C6MH3-B47, C6 MH3-B46, C6 MH3-B43, C6 MH3-B41, C6 MH3-B39, C6 MH3-B34, C6MH3-B33, C6 MH3-B31, C6 MH3-B27, C6 MH3-B25, C6 MH3-B21, C6 MH3-B20, C6MH3-B2, C6 MH3-B16, C6 MH3-B15, C6 MH3-B11, C6 MH3-B1, C6 MH3-A3, C6MH3-A2, and C6ML3-9. These and other anti-HER2/neu antibodies aredescribed in U.S. Pat. Nos. 6,512,097 and 5,977,322, in PCT PublicationWO 97/00271, in Schier et al. (1996) J Mol Blot 255: 28-43, Schier etal. (1996) J Mol Biol 263: 551-567, and the like.

More generally, antibodies directed to various members of the epidermalgrowth factor receptor family are well suited for use as targetingmoieties in the constructs of the present invention. Such antibodiesinclude, but are not limited to anti-EGF-R antibodies as described inU.S. Pat. Nos. 5,844,093 and 5,558,864, and in European Patent No.706,799A). Other illustrative anti-EGFR family antibodies include, butare not limited to antibodies such as C6.5, C6ML3-9, C6 MH3-B1, C6-B1D2,F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12,EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8,HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8and HER4.C7 and the like (see, e.g., U.S. Patent publications US2006/0099205 A1 and US 2004/0071696 A1 which are incorporated herein byreference).

As described in U.S. Pat. Nos. 6,512,097 and 5,977,322 other anti-EGFRfamily member antibodies can readily be produced by shuffling lightand/or heavy chains followed by one or more rounds of affinityselection. Thus in certain embodiments, this invention contemplates theuse of one, two, or three CDRs in the VL and/or VH region that are CDRsdescribed in the above-identified antibodies and/or the above identifiedpublications.

In various embodiments the targeting moiety comprises an antibody thatspecifically or preferentially binds CD20. Anti-CD20 antibodies are wellknown to those of skill and include, but are not limited to rituximab,ibritumomab tiuxetan, and tositumomab, AME-133v (Applied MolecularEvolution), Ocrelizumab (Roche), Ofatumumab (Genmab), TRU-015 (Trubion)and IMMU-106 (Immunomedics).

The invention need not be limited to the use of the antibodies describedabove, and other such antibodies as they are known to those of skill inthe art can be used in the compositions and methods described herein.

While the above discussion pertains to antibodies, it will be recognizedthat affybodies and/or unibodies can be used instead of antibodies.

Unibodies.

UniBody are antibody technology that produces a stable, smaller antibodyformat with an anticipated longer therapeutic window than certain smallantibody formats. In certain embodiments unibodies are produced fromIgG4 antibodies by eliminating the hinge region of the antibody. Unlikethe full size IgG4 antibody, the half molecule fragment is very stableand is termed a uniBody. Halving the IgG4 molecule left only one area onthe UniBody that can bind to a target. Methods of producing unibodiesare described in detail in PCT Publication WO2007/059782, which isincorporated herein by reference in its entirety (see, also, Kolfschotenet al. (2007) Science 317: 1554-1557).

Affibodies.

Affibody molecules are class of affinity proteins based on a 58-aminoacid residue protein domain, derived from one of the IgG-binding domainsof staphylococcal protein A. This three helix bundle domain has beenused as a scaffold for the construction of combinatorial phagemidlibraries, from which Affibody variants that target the desiredmolecules can be selected using phage display technology (see, e.g.,Nord et al. (1997) Nat. Biotechnol. 15: 772-777; Ronmark et al. (2002)Eur. J. Biochem., 269: 2647-2655). Details of Affibodies and methods ofproduction are known to those of skill (see, e.g., U.S. Pat. No.5,831,012 which is incorporated herein by reference in its entirety).

It will be recognized that the antibodies described above can beprovided as whole intact antibodies (e.g., IgG), antibody fragments, orsingle chain antibodies, using methods well known to those of skill inthe art. In addition, while the antibody can be from essentially anymammalian species, to reduce immunogenicity, it is desirable to use anantibody that is of the species in which the construct (e.g.,anti-HER2/neu-IFN-α chimera) is to be used. In other words, for use in ahuman, it is desirable to use a human, humanized, or chimeric humanantibody.

B) Interferons

In various embodiments chimeric moieties of this invention comprise aninterferon (e.g., IFN-α, IFN-β, etc.) joined to the targeting moiety(e.g., anti-HER2/neu antibody). The interferon can be a full lengthwild-type interferon (e.g. IFN-α, IFN-β, IFN-γ, etc.) an interferonfragment (e.g., an IFN-α fragment), and/or a mutated interferon.Typically the interferon fragment is one that possesses the endogenousactivity of the native interferon, preferably at a level of at least80%, more preferably at least 90% or 95%, most preferably at least 98%,99%, 100%, or a level greater than the wild-type interferon.

Means of identifying such modified interferon molecules are routine tothose of skill in the art. In one illustrative approach, a library oftruncated and/or mutated IFN-α is produced and screened for IFN-αactivity. Methods of producing libraries of polypeptide variants arewell known to those of skill in the art. Thus, for example error-pronePCR can be used to create a library of mutant and/or truncated IFN-α(see, e.g., U.S. Pat. No. 6,365,408).

The resulting library members can then be screened according to standardmethods know to those of skill in the art. Thus, for example, IFN-αactivity can be assayed by measuring antiviral activity against aparticular test virus. Kits for assaying for IFN-α activity arecommercially available (see, e.g., ILITE™ alphabeta kit by Neutekbio,Ireland).

In various embodiments use of a mutated interferon alpha 2 (IFNα2) iscontemplated. Certain mutants include a mutation of the H is at position57, and/or the E at position 58, and/or the Q at position 61. In certainembodiments the mutants include the mutation H57Y, and/or E58N, and/orQ61S. In certain embodiments the mutants include a mutated IFNα2 havingthe mutations H57Y, E58N, and Q61S (YNS) (see, e.g., Kalie et al. (2007)J. Biol. Chem., 282: 11602-11611).

A mutated IFN-β comprising a serine substituted for the naturallyoccurring cysteine at amino acid 17 has also been demonstrated to showefficacy (see, e.g., Hawkins et al. (1985) Cancer Res., 45, 5914-5920.

In various embodiments use of truncated interferons is alsocontemplated. Human INFα, for example, with deletions of the first 15amino-terminal amino acid residues and/or the last 10-13carboxyl-terminal amino acid residues, have been shown to exhibitvirtually the same activity as the parent molecules (see, e.g., Ackerman(1984) Proc. Natl. Acad. Sci., USA, 81: 1045-1047). Accordingly the useof IFN-αs having 1, 2, 3, up to 13 carobxyl terminal amino acid residuesdeleted and/or 1, 2, 3, up to 15 amino terminal amino acid residuesdeleted are contemplated.

It has also been demonstrated that activity resides in huIFN-α fragmentHuIFN-α (1-110) (Id.). Accordingly carboxyl truncated IFNs withtruncations after residue 110 and/or with 1, 2, 3, up to 15 aminoterminal amino acid residues deleted are contemplated.

Certain C-terminally truncated interferon betas (IFN-β) have been shownto have increased activity (see, e.g., U.S. Patent Publication2009/0025106 A1). Accordingly, in certain embodiments the interferonsused in the constructs described herein include the C-terminallytruncated IFN-β described as IFN-Δ1, IFN-Δ2, IFN-Δ3, IFN-Δ4, IFN-Δ5,IFN-Δ6, IFN-Δ7, IFN-Δ8, IFN-Δ9, IFN-Δ10 in US 2009/0025106 A1. Incertain embodiments the interferon is IFN-Δ7, IFN-Δ8, IFN-Δ9 (SEQ IDNOs: 57, 59, and 61 in US 2009/0025106 A1 (see, Table 3).

TABLE 3 Truncated IFN-βshowing enhanced activity (see U.S. patent Publication 2009/0025106 A1).SEQ Truncated ID IFN Amino Acid Sequence NO IFN-Δ7Met Gly Lys Met Ala Ser Leu Phe Ala Thr Phe Leu Val Val Leu Val 50Ser Leu Ser Leu Ala Ser Glu Ser Ser Ala Cys Asp Leu Pro Gln ThrHis Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met ArgArg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly PhePro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile ProVal Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr LysAsp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr ThrGlu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln GlyVal Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu AlaVal Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys LysTyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg SerPhe Ser Leu Ser Thr Asn Leu Gln IFN-Δ8Met Gly Lys Met Ala Ser Leu Phe Ala Thr Phe Leu Val Val Leu Val 51Ser Leu Ser Leu Ala Ser Glu Ser Ser Ala Cys Asp Leu Pro Gln ThrHis Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met ArgArg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly PhePro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile ProVal Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr LysAsp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr ThrGlu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln GlyVal Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu AlaVal Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys LysTyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg SerPhe Ser Leu Ser Thr Asn Leu IFN-Δ9Met Gly Lys Met Ala Ser Leu Phe Ala Thr Phe Leu Val Val Leu Val 52Ser Leu Ser Leu Ala Ser Glu Ser Ser Ala Cys Asp Leu Pro Gln ThrHis Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met ArgArg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly PhePro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile ProVal Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr LysAsp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr ThrGlu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln GlyVal Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu AlaVal Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys LysTyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg SerPhe Ser Leu Ser Thr Asn

The use of chemically modified interferons is also contemplated. Forexample, in certain embodiments, the interferon is chemically modifiedto increase serum half-life. Thus, for example,(2-sulfo-9-fluorenylmethoxycarbonyl)₇-interferon-α2 undergoestime-dependent spontaneous hydrolysis, generating active interferon(see, e.g., Shechter et al. (2001) Proc. Natl. Acad. Sci., USA, 98(3):1212-1217). Other modifications, include for example, N-terminalmodifications in including, but not limited to the addition of PEG,protecting groups, and the like. U.S. Pat. No. 5,824,784, for example,described N-terminally chemically modified interferon.

The foregoing interferons are intended to be illustrative and notlimiting. Using the teaching provided herein, other suitable modifiedinterferons (e.g., modified IFN-α, IFN-β, IFN-γ, etc.) can readily beidentified and produced.

C. Attachment of the Antibody (e.g., Anti-HER2/Neu) to the IFN-α.

Generally speaking, the targeting moiety (e.g., an anti-HER2/neuantibody, and anti-CD20 antibody, etc.) can be joined together in anyorder. Thus, for example, the antibody can be joined to either the aminoor carboxy terminal of the interferon. The antibody can also be joinedto an internal region of the interferon, or conversely, the interferoncan be joined to an internal location or to any terminus of theantibody, as long as the attachment does not interfere with binding ofthe antibody to that target marker (e.g., the HER2/neu receptor).

The antibody (e.g., a C6 anti-HER2/neu, anti-CD20, etc.) and theinterferon (e.g., IFN-α, IFN-β, etc.) can be attached by any of a numberof means well known to those of skill in the art. In certainembodiments, the interferon is conjugated, either directly or through alinker (spacer), to the antibody. In certain embodiments, however, it ispreferable to recombinantly express the chimeric moiety as a fusionprotein.

i) Chemical Conjugation of the Targeting Moiety to the Interferon.

In certain embodiments, the targeting moiety (e.g., an anti-CD20antibody such as rituximab, an anti-HER2/neu antibody such as C6.5, C6MH3-B1, G98A, ML3-9, H3B1, B1D2, etc.) is chemically conjugated to theinterferon (e.g., IFN-α, IFN-β, etc.) molecule. Means of chemicallyconjugating molecules are well known to those of skill.

The procedure for conjugating two molecules varies according to thechemical structure of the agent. Polypeptides typically contain varietyof functional groups; e.g., carboxylic acid (COOH) or free amine (—NH₂)groups, that are available for reaction with a suitable functional groupon the other peptide, or on a linker to join the molecules thereto.

Alternatively, the antibody and/or the interferon can be derivatized toexpose or attach additional reactive functional groups. Thederivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, typically refers to a molecule that is usedto join the antibody to the interferon. In various embodiments, thelinker is capable of forming covalent bonds to both the antibody and tothe interferon. Suitable linkers are well known to those of skill in theart and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Incertain embodiments, the linker(s) can be joined to the constituentamino acids of the antibody and/or the interferon through their sidegroups (e.g., through a disulfide linkage to cysteine). In certainpreferred embodiments, the linkers are joined to the alpha carbon aminoand/or carboxyl groups of the terminal amino acids of the antibodyand/or the interferon.

A bifunctional linker having one functional group reactive with a groupon the antibody and another group reactive on the interferon, can beused to form the desired conjugate. Alternatively, derivatization caninvolve chemical treatment of the targeting moiety. Procedures forgeneration of, for example, free sulfhydryl groups on polypeptides, suchas antibodies or antibody fragments, are known (See U.S. Pat. No.4,659,839).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known. See, for example, European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075. In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; U.S. Pat.Nos. 4,545,985 and 4,894,443, and the like.

ii) Production of Fusion Proteins.

In certain embodiments, a chimeric targeting moiety-interferon fusionprotein is synthesized using recombinant DNA methodology. Generally thisinvolves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins (e.g. anti-HER2/neu-IFN-α,anti-HER2/neu-IFN-β, anti-CD20-IFN-α, anti-CD20-IFN-β, etc.) describedherein can be prepared by any suitable method, including, for example,cloning and restriction of appropriate sequences or direct chemicalsynthesis by methods such as the phosphotriester method of Narang et al.(1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown etal. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite methodof Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862); the solidsupport method of U.S. Pat. No. 4,458,066, and the like.

Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments can then be ligated to produce the desired DNA sequence.

In certain embodiments, DNA encoding fusion proteins of the presentinvention can be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the gene for theinterferon (e.g., IFN-α) is PCR amplified, using a sense primercontaining the restriction site for, e.g., NdeI and an antisense primercontaining the restriction site for HindIII. This can produce a nucleicacid encoding the mature interferon sequence and having terminalrestriction sites. An antibody having “complementary” restriction sitescan similarly be cloned and then ligated to the interferon and/or to alinker attached to the interferon. Ligation of the nucleic acidsequences and insertion into a vector produces a vector encoding theinterferon joined to the antibody (e.g., anti-CD20).

While the two molecules can be directly joined together, one of skillwill appreciate that the molecules can be separated by a peptide spacerconsisting of one or more amino acids. Generally the spacer will have nospecific biological activity other than to join the proteins or topreserve some minimum distance or other spatial relationship betweenthem. In certain embodiments, however, the constituent amino acids ofthe spacer can be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

It was a surprising discovery, however, that certain linkers areunsuitable for preparation of fusion proteins of the present invention.Thus, for example, the (Gly₄Ser)₃ (SEQ ID NO:5) linker was not wellsuited for the production of an anti-CD20-IFN construct. Without beingbound to a particular theory, it is believed the interferon was beingremoved from the fusion protein by proteolysis. Western blot analysisusing anti-Fc and anti-interferon, confirmed that both of the upperbands were heavy chains, but only the largest contained interferon.

It was also a surprising discovery that proteolysis resistant linkersand in certain embodiments, “short” proteolysis resistant linkersproduced a targeted interferon construct that had greater activity (evenin vitro) against cells expressing the target moiety (e.g., CD20) thanan untargeted construct.

Accordingly, in certain preferred embodiments, it is desirable to use alinker that is resistant to proteolysis. Certain preferred linkers arelinkers that are not the (Gly₄Ser)₃ (SEQ ID NO:5) linker. Certainpreferred linkers are linkers shorter than 15 amino acids, or linkersshorter than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acidsin length. In certain embodiments the linker is an alpha helical linkerranging in length up to about 12 or 13 or 14 amino acids in length. Incertain embodiments the linker is a Gly₄Ser (SEQ ID NO:6) linker or alinker approximately equal to in length or shorter than a (Gly₄Ser)₂(SEQ ID NO:53) more preferably a linker approximately equal to in lengthor shorter than a Gly₄Ser (SEQ ID NO:6) linker.

Certain illustrative proteolysis-resistant linkers well suited for usein the constructs of this invention are shown in Table 4.

TABLE 4 Illustrative proteolysis-resistant linkers. Linker Seq SEQ ID NOGGGGS 6 GGGGSGGGGS 53 AEAAAKEAAAKA 7 A(EAAAK)_(n)A where n = 1, 54where n = 2 55 where n = 3 56 where n = 4, 57 where n = 5 58 GGGGG 59GGGGGGGG 60 GGAGG 61 GAGAGAGAGA 62 RPLSYRPPFPFGFPSVRP 63YPRSIYIRRRHPSPSLTT 64 TPSHLSHILPSFGLPTFN 65 RPVSPFTFPRLSNSWLPA 66SPAAHFPRSIPRPGPIRT 67 APGPSAPSHRSLPSRAFG 68 PRNSIHFLHPLLVAPLGA 69MPSLSGVLQVRYLSPPDL 70 SPQYPSPLTLTLPPHPSL 71 NPSLNPPSYLHRAPSRIS 72LPWRTSLLPSLPLRRRP 73 PPLFAKGPVGLLSRSFPP 74 VPPAPVVSLRSAHARPPY 75LRPTPPRVRSYTCCPTP 76 PNVAHVLPLLTVPWDNLR 77 CNPLLPLCARSPAVRTFP 78

It was also a surprising discovery, as illustrated in FIG. 29anti-CD20-IFN showed a dramatic gain in tumor specific potency ascompared to IFN and anti-DNS-IFN, even in vitro. Interestingly, bothantibody-IFN fusion molecules (anti-CD20-IFN and anti-DNS-IFN) showedlower potency than unfused IFN as depicted in the left panel of thefigure. Without being bound to a particular theory, it is believed thisis due to the shorter linker causing a steric hindrance. In certainembodiments the targeting moiety-interferon construct shows at least1.25, preferably at least 1.5, more preferably at least 2×, still morepreferably at least 5×, 10×, 20×, 50×, or at least 100× greater activitythan the corresponding interferon without a targeting moiety.

The nucleic acid sequences encoding the fusion proteins can be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene istypically operably linked to appropriate expression control sequencesfor each host. For E. coli this includes a promoter such as the T7, trp,or lambda promoters, a ribosome binding site and preferably atranscription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.: Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y., and the like). Substantially pure compositions of at least about90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneityare most preferred for pharmaceutical uses. Once purified, partially orto homogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the fusion protein (e.g.,anti-HER2/neu-IFN-α, anti-CD20-IFN-α, etc.) may possess a conformationsubstantially different than the native conformations of the constituentpolypeptides. In this case, it may be necessary to denature and reducethe polypeptide and then to cause the polypeptide to re-fold into thepreferred conformation. Methods of reducing and denaturing proteins andinducing re-folding are well known to those of skill in the art (see,e.g., Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitmanand Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al.(1992) Anal. Biochem., 205: 263-270). Debinski et al., for example,describe the denaturation and reduction of inclusion body proteins inguanidine-DTE. The protein is then refolded in a redox buffer containingoxidized glutathione and L-arginine.

In certain embodiments a transient expression system can be used toexpress the chimeric constructs described herein. Although many celllines potentially can be used, one cell line that works well fortransient expression is 293T. For transient expression of 293T on Day 0,9 million cells in 25 ml are seeded for each 150 mm tissue cultureplate. A 1 mg/ml of PEI (Polyethylenimine) is made using sterile water.For the expression of a complete antibody or antibody fusion protein, 25μg each of H and L (50 ug total) is used per plate. A volume of 5 ml isused for transfection of each 150 mm plate. The DNA is mixed with DMEM,the PEI is then added and the mixture is incubated at room temperaturefor 10 mins. 1.75 μg PEI is used for each ug of DNA. For transfection,the old medium is removed, discarded and replaced with 20 ml of freshmedium (Iscoves+5% calf serum). The transfection mix is added and theplate is swirled. On Day 2, the medium is replaced with 30 ml of Iscovesmedium containing 1% FBS (fetal bovine serum) to minimize the amount ofbovine Ig present. Supernatants are collected from the cells on Days 4,6 and 13 by removing the medium and replacing it with 30 ml of freshIscoves containing 1% FBS.

The cloning and expression of an anti-HER2/neu-IFN-α fusion protein isillustrated herein in Example 1, while the cloning and expression of ananti-CD20-IFN-α fusion protein is shown in Example 2.

One of skill would recognize these expression methods are illustrativeand not limiting. Modifications can be made to the fusion proteinsdescribed herein without diminishing their activity/efficacy. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

Other modifications can be made to increase serum half-life and/orbioavailability. Such modifications include, but are not limited to theincorporation of D amino acids (especially in the linker), the use ofnon-naturally occurring amino acids, pegylation of the fusion protein,and the like.

D. Other Multi-Valent Targeting Moieties.

In certain embodiments this invention contemplates the use ofmultivalent, preferably trivalent, quadravalent, pentavalent or greatertargeting moieties (e.g., anti-HER2/neu antibodies, anti-CD20antibodies, etc.) to target the interferon to a target cell.

For example, multivalent anti-HER2/neu moieties can be produced by anyof a number of methods. For example, linkers having three, four, or morereactive sites can be reacted with anti-HER2/neu antibodies to form atrimer or greater conjugate.

In certain embodiments, phage display, yeast display, bacterial display,or other display systems can be used to express and display multiplecopies (e.g., at least 3, at least 4, at least 5, at least 6 copies,etc.) of a targeting (e.g., anti-HER2/neu, anti-CD20, etc.) antibody andthereby effectively provide a multivalent targeting moiety.

In certain embodiments the use of diabodies and triabodies (e.g.,comprising two domains that bind CD-20 or one domain that binds CD20 andanother domain that binds, for example, a different member of the EGFRreceptor family (e.g., EGFR, HER3, etc.). Typically, diabodies comprisea heavy (VH) chain variable domain connected to a light chain variabledomain (VL) on the same polypeptide chain (V_(H)-V_(L)) connected by apeptide linker that is too short to allow pairing between the twodomains on the same chain. This forces pairing with the complementarydomains of another chain and promotes the assembly of a dimeric moleculewith two functional antigen binding sites (see, e.g., Holliger et al.(1993) Proc. Natl. Acad. Sci., 90: 6444-6448). In certain embodiments toconstruct bispecific diabodies the V-domains of antibody A and antibodyB are fused to create the two chains VHA-VLB, VHB-VLA. Each chain isinactive in binding to antigen, but recreates the functional antigenbinding sites of antibodies A and B on pairing with the other chain.

II. Combined Uses.

The chimeric constructs of this invention are useful for inhibiting thegrowth and/or proliferation of target cells (e.g., cancer cells). Invarious embodiments the chimeric moieties can be used to inhibit diseaseprogression, to shrink tumor size, and/or to stabilizeregression/remission.

Particularly, in the treatment of cancer, the compositions and methodsof the invention may also include additional therapeutic and/orpharmacologically acceptable agents. For instance, the compositions ormethods may involve other agents for the treatment of cancer. Suchagents include, but are not limited to alkylating agents (e.g.,mechlorethamine (Mustargen), cyclophosphamide (Cytoxan, Neosar),ifosfamide (Ifex), phenylalanine mustard; melphalen (Alkeran),chlorambucol (Leukeran), uracil mustard, estramustine (Emcyt), thiotepa(Thioplex), busulfan (Myerlan), lomustine (CeeNU), carmustine (BiCNU,BCNU), streptozocin (Zanosar), dacarbazine (DTIC-Dome), cis-platinum,cisplatin (Platinol, Platinol AQ), carboplatin (Paraplatin), altretamine(Hexylen), etc.), antimetabolites (e.g. methotrexate (Amethopterin,Folex, Mexate, Rheumatrex), 5-fluoruracil (Adrucil, Efudex, Fluoroplex),floxuridine, 5-fluorodeoxyuridine (FUDR), capecitabine (Xeloda),fludarabine: (Fludara), cytosine arabinoside (Cytaribine, Cytosar,ARA-C), 6-mercaptopurine (Purinethol), 6-thioguanine (Thioguanine),gemcitabine (Gemzar), cladribine (Leustatin), deoxycoformycin;pentostatin (Nipent), etc.), antibiotics (e.g. doxorubicin (Adriamycin,Rubex, Doxil, Daunoxome-liposomal preparation), daunorubicin(Daunomycin, Cerubidine), idarubicin (Idamycin), valrubicin (Valstar),mitoxantrone (Novantrone), dactinomycin (Actinomycin D, Cosmegen),mithramycin, plicamycin (Mithracin), mitomycin C (Mutamycin), bleomycin(Blenoxane), procarbazine (Matulane), etc.), mitotic inhibitors (e.g.paclitaxel (Taxol), docetaxel (Taxotere), vinblatine sulfate (Velban,Velsar, VLB), vincristine sulfate (Oncovin, Vincasar PFS, Vincrex),vinorelbine sulfate (Navelbine), etc.), chromatin function inhibitors(e.g., topotecan (Camptosar), irinotecan (Hycamtin), etoposide (VP-16,VePesid, Toposar), teniposide (VM-26, Vumon), etc.), hormones andhormone inhibitors (e.g. diethylstilbesterol (Stilbesterol,Stilphostrol), estradiol, estrogen, esterified estrogens (Estratab,Menest), estramustine (Emcyt), tamoxifen (Nolvadex), toremifene(Fareston) anastrozole (Arimidex), letrozole (Femara),17-OH-progesterone, medroxyprogesterone, megestrol acetate (Megace),goserelin (Zoladex), leuprolide (Leupron), testosteraone,methyltestosterone, fluoxmesterone (Android-F, Halotestin), flutamide(Eulexin), bicalutamide (Casodex), nilutamide (Nilandron), etc.),inhibitors of synthesis (e.g., aminoglutethimide (Cytadren),ketoconazole (Nizoral), etc.), immunomodulators (e.g., rituximab(RITUXAN®), trastuzumab (Herceptin), denileukin diftitox (Ontak),levamisole (Ergamisol), bacillus Calmette-Guerin, BCG (TheraCys, TICEBCG), interferon alpha-2a, alpha 2b (Roferon-A, Intron A),interleukin-2, aldesleukin (ProLeukin), etc.) and other agents such as1-aspariginase (Elspar, Kidrolase), pegaspasgase (Oncaspar), hydroxyurea(Hydrea, Doxia), leucovorin (Wellcovorin), mitotane (Lysodren), porfimer(Photofrin), tretinoin (Veasnoid), and the like.

III. Pharmaceutical Compositions.

In order to carry out the methods of the invention, one or more activeagents (chimeric moieties) of this invention are administered, e.g. toan individual diagnosed as having a cancer. The active agent(s) can beadministered in the “native” form or, if desired, in the form of salts,esters, amides, prodrugs, derivatives, and the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologically, i.e.,effective in the present method. Salts, esters, amides, prodrugs andother derivatives of the active agents can be prepared using standardprocedures known to those skilled in the art of synthetic organicchemistry and described, for example, by March (1992) Advanced OrganicChemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y.Wiley-Interscience.

For example, acid addition salts are prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the active agents of thisinvention are prepared in a similar manner using a pharmaceuticallyacceptable base such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimethylamine, or the like. Particularlypreferred basic salts include alkali metal salts, e.g., the sodium salt,and copper salts.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups which may be present within the molecularstructure of the drug. The esters are typically acyl-substitutedderivatives of free alcohol groups, i.e., moieties that are derived fromcarboxylic acids of the formula RCOOH where R is alky, and preferably islower alkyl. Esters can be reconverted to the free acids, if desired, byusing conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs can also be prepared using techniques known to thoseskilled in the art or described in the pertinent literature. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

The active agents identified herein are useful for parenteral, topical,oral, nasal (or otherwise inhaled), rectal, or local administration,such as by aerosol or transdermally, for prophylactic and/or therapeutictreatment of one or more of the pathologies/indications described herein(e.g., atherosclerosis and/or symptoms thereof). The pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. Suitable unit dosage forms,include, but are not limited to powders, tablets, pills, capsules,lozenges, suppositories, patches, nasal sprays, injectables, implantablesustained-release formulations, lipid complexes, etc.

The active agents of this invention are typically combined with apharmaceutically acceptable carrier (excipient) to form apharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well-knownsterilization techniques.

In therapeutic applications, the compositions of this invention areadministered to a patient suffering e.g. from a cancer, or at risk ofcancer (e.g. after surgical removal of a primary tumor) in an amountsufficient to prevent and/or cure and/or or at least partially preventor arrest the disease and/or its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. Single ormultiple administrations of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the active agents of the formulations of this invention toeffectively treat (ameliorate one or more symptoms) the patient.

The concentration of active agent(s) can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs. Concentrations, however, willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosagesrange from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably fromabout 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferredembodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. In certain embodiments, dosages range from about 20 mg toabout 50 mg given orally twice daily. It will be appreciated that suchdosages may be varied to optimize a therapeutic regimen in a particularsubject or group of subjects.

In certain preferred embodiments, the active agents of this inventionare administered orally (e.g. via a tablet) or as an injectable inaccordance with standard methods well known to those of skill in theart. In other preferred embodiments, the peptides, may also be deliveredthrough the skin using conventional transdermal drug delivery systems,i.e., transdermal “patches” wherein the active agent(s) are typicallycontained within a laminated structure that serves as a drug deliverydevice to be affixed to the skin. In such a structure, the drugcomposition is typically contained in a layer, or “reservoir,”underlying an upper backing layer. It will be appreciated that the term“reservoir” in this context refers to a quantity of “activeingredient(s)” that is ultimately available for delivery to the surfaceof the skin. Thus, for example, the “reservoir” may include the activeingredient(s) in an adhesive on a backing layer of the patch, or in anyof a variety of different matrix formulations known to those of skill inthe art. The patch may contain a single reservoir, or it may containmultiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

In certain embodiments elevated serum half-life can be maintained by theuse of sustained-release protein “packaging” systems. Such sustainedrelease systems are well known to those of skill in the art. In onepreferred embodiment, the ProLease™ biodegradable microsphere deliverysystem for proteins and peptides (see, e.g., Tracy (1998) Biotechnol.Prog. 14: 108; Johnson et al. (1996), Nature Med. 2: 795; Herbert et al.(1998), Pharmaceut. Res. 15, 357) a dry powder composed of biodegradablepolymeric microspheres containing the active agent in a polymer matrixthat can be compounded as a dry formulation with or without otheragents.

The ProLease™ microsphere fabrication process was specifically designedto achieve a high encapsulation efficiency while maintaining integrityof the active agent. The process consists of (i) preparation offreeze-dried drug particles from bulk by spray freeze-drying the drugsolution with stabilizing excipients, (ii) preparation of a drug-polymersuspension followed by sonication or homogenization to reduce the drugparticle size, (iii) production of frozen drug-polymer microspheres byatomization into liquid nitrogen, (iv) extraction of the polymer solventwith ethanol, and (v) filtration and vacuum drying to produce the finaldry-powder product. The resulting powder contains the solid form of theactive agents, which is homogeneously and rigidly dispersed withinporous polymer particles. The polymer most commonly used in the process,poly(lactide-co-glycolide) (PLG), is both biocompatible andbiodegradable.

Encapsulation can be achieved at low temperatures (e.g., −40° C.).During encapsulation, the protein is maintained in the solid state inthe absence of water, thus minimizing water-induced conformationalmobility of the protein, preventing protein degradation reactions thatinclude water as a reactant, and avoiding organic-aqueous interfaceswhere proteins may undergo denaturation. A preferred process usessolvents in which most proteins are insoluble, thus yielding highencapsulation efficiencies (e.g., greater than 95%).

In another embodiment, one or more components of the solution can beprovided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

IV. Kits.

In certain embodiments, this invention provides for kits for thetreatment a primary cancer and/or in an adjunct therapy. Kits typicallycomprise a container containing a chimeric moiety of the presentinvention (e.g., anti-HER2/neu-IFN-α, anti-CD20-IFN-α, etc.). Thechimeric moiety can be present in a pharmacologically acceptableexcipient.

In addition the kits can optionally include instructional materialsdisclosing means of use of the chimeric moiety (e.g. to treat a cancerand/or as an adjunct therapeutic). The instructional materials may also,optionally, teach preferred dosages, counter-indications, and the like.

The kits can also include additional components to facilitate theparticular application for which the kit is designed. Thus, for example,and additionally comprise means for disinfecting a wound, for reducingpain, for attachment of a dressing, and the like.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Anti-HER2/Neu IgG3 and IFN-Alpha Fusion Protein DemonstratesPotent Apoptotic and Anti-Tumor Activities Against B Cell Lymphoma

In the present study, we constructed a fusion protein consisting ofanti-HER2/neu-IgG3 with the variable region of C6 MH3-B1 (Schier et al.(1996) J. Mol. Biol. 263: 551-567) and IFN-α, and investigated itseffect on a murine B cell lymphoma, 38C13, expressing human HER2/neu(38C13/HER2). We chose to evaluate IFN-α targeting to tumor in thismodel given the responsiveness of this B cell lymphoma to IFN-α (Reid etal. (1989) Cancer Res. 49: 4163-4169). Fusion of IFN-α to an Absignificantly increased its in vivo half-life. Anti-HER2/neu-IgG3-IFN-αwas found to be efficient in inhibiting the growth in vivo of both smalland established 38C13/HER2 tumors with no signs of systemic toxicityobserved at effective doses. Anti-HER2/neu-IgG3-IFN-α inhibited thegrowth of and induced apoptosis in 38C13/HER2 cells. These resultsindicate that fusion of IFN-α to a tumor-specific antibody results in anagent effective for the treatment of B cell lymphoma.

Materials and Methods

Cell Lines and Culture Conditions

38C13 is a highly malignant murine B cell lymphoma derived from C3H/HeNmice. The construction and characterization of 38C13 expressing humanHER2/neu (38C13/HER2) has been previously described (Dela Cruz et al.(2000) Immunol. 165: 5112-5121). Both 38C13 and 38C13/HER2 were culturedin IMDM (Irvine Scientific) supplemented with 2 mM L-glutamine, 10 U/mlpenicillin, 10 microg/ml streptomycin (GPS; Sigma-Aldrich) and 10% calfserum (Atlanta Biologicals). Murine myeloma P3X63Ag8.653 (American TypeCulture Collection) and its derivatives expressing anti-HER2 IgG3-IFN-αor IgG3-IFN-α were grown in IMDM supplemented with 10% calf serum andGPS. L929 fibroblasts (American Type Culture Collection) were culturedin IMDM with 5% calf serum and GPS. The construction andcharacterization of CT26/HER2, a murine colon adenocarcinoma cell lineoverexpressing human HER2/neu, has been previously described (Id.).CT26/HER2 was cultured in IMDM with 5% calf serum and GPS.

Plasmid Construction

The H and L chain variable regions of C6 MH3-B1, an anti-human HER2/neuscFv were inserted into the human γ3 H chain (pAH4802) and κL chain(pAG4622) expression vectors, respectively (Coloma et al. (1992)Immunol. Methods 152: 89-104), and used to produce chimeric IgG3 of thisspecificity. To construct the anti-human HER2/neu-IgG3(C6 MH3-B1)-IFN-αfusion protein, PCR was first used to introduce a BamH1 restrictionenzyme site upstream and XbaI restriction enzyme site downstream of themature murine IFN-α gene amplified by PCR from genomic DNA of BALB/cmice with the forward primer 5′-CGC GGA TCC TGT GAC CTG CCT CAG ACT C-3(SEQ ID NO:79) and the reverse primer 5′-GCT CTA GAT CAT TTC TCT TCT CTCAGT CTT C-3 (SEQ ID NO:80). The final PCR product was ligated into a TAvector. The resulting vector, after sequencing, was digested with BamH1and XbaI to release the DNA fragment which was inserted into the vectorpAH9612 containing the IgG3 constant region with the C6 MH3-B1 H chainvariable region and a GGGGSGGGGSGGGGS (SEQ ID NO:5) peptide linker atthe end of C_(H)3. The final PCR product, pAH9616, containedanti-HER2/neu-IgG3 followed by a GGGGSGGGGSGGGGS (SEQ ID NO: 5) peptidelinker and murine IFN-α.

Production and Purification of Recombinant Proteins

Plasmid encoding the IgG3 H chain with the C6 MH3-B1 variable regionfused to IFN-α was transfected into P3X63Ag8.653 cells expressing eitherL chain with the C6 MH3-B1 variable region (Huang and Morrison (2005) J.Pharmacol. Exp. Ther. 316: 983-991) to produce anti-HER2/neu-IgG3-IFN-αor nonspecific L chain (4D5; Genentech) (Dela Cruz et al. (2000)Immunol. 165: 5112-5121) to produce IgG3-IFN-α by electroporation with apulse of 960 μFd capacitance and 0.2 V. Transfectants producinganti-HER2/neu(C6 MH3-B1)-IgG3, anti-HER2/neu(C6 MH3-B1)-IgG3-IFN-α, orIgG3-IFN-α were selected and characterized as previously described(Id.). Anti-HER2/neu(C6 MH3-B1)-IgG3 was purified from culturesupernatants using protein G immobilized on Sepharose 4B fast flow(Sigma-Aldrich), and anti-HER2/neu(C6 MH3-B1)-IgG3-IFN-α and IgG3-IFN-αwere purified from culture supernatants using protein A immobilized onSepharose 4B fast flow (Sigma-Aldrich). Purity and integrity wereassessed by Coomassie blue staining of proteins separated by SDS-PAGE.The international reference standard for mouse IFN-α provided by theNational Institutes of Health was used to determine IFN activity of thefusion proteins. rIFN-α was obtained from PBL Biomedical Laboratories.

FPLC Analysis of IgG3-IFN-α Fusion Protein

To determine whether the fusion protein exists as monomer and/orpolymers in solution, 100 μg of IgG3-IFN-α mixed with 400 μg of OVA toprovide an internal control was analyzed by gel filtration on a30×1.5-cm Superose 6 column attached in a fast protein liquidchromatography (FPLC) using PBS and 0.5 ml/min flow rate. Gel filtrationon the same column of IgA2m that exists predominantly as dimer Ab with amolecular mass of 350 kDa and a mixture of Miles IgG of molecular mass150 kDa and OVA of molecular mass 45 kDa were used to provide molecularmass standards.

Flow Cytometry Analysis of HER2/Neu-Binding Activity

To detect the reactivity of various anti-HER2/neu fusion proteins withCT26/HER2 cells, 1×10⁶ cells were incubated at 4° C. for 1 h with 10 pMof the fusion protein. For some experiments, the fusion proteins werepreincubated with 900 U of heparin at 4° C. for 17 h before incubationwith CT26/HER2 cells. Cells were then reacted with biotinylated ratanti-human IgG (BD Biosciences) diluted 1/100. The bound biotinylatedAbs were detected with PE-labeled streptavidin (BD Biosciences) diluted1/1500 and cells were analyzed by flow cytometry using a FACScan (BDBiosciences).

IFN-α Antiviral Activity

The L-929 fibroblast cell line sensitive to the vesicular stomatitisvirus (VSV) infection was used to quantify the biological activity ofIFN-α. L-929 cells were plated in a 96-well tissue culture plate(Falcon; BD Biosciences) at a density of 4×10⁴ cells/well and incubatedovernight at 37° C. in a 5% CO2 atmosphere. Afterward, serial dilutionsof different IFN-α fusion proteins or standard IFN-α (internationalreference standard for mouse IFN-α; National Institutes of Health,Bethesda, Md.) were added and the plate was incubated at 37° C. for 24h. Four thousand PFU of VSV was then added to each well and incubated at37° C. for another 48 h. Surviving adherent cells were stained with 50μl of crystal violet (0.05% in 20% ethanol) for 10 min. The plates werewashed with water and the remaining dye was solubilized by the additionof 100 μl of 100% methanol. Plates were read using an ELISA reader at595 nm.

Assay for the Antiproliferative Effect of Anti-HER2/Neu-IgG3-IFN-α

In brief, 38C13 or 38C13/HER2 cells were plated in a 96-well tissueculture plate at a density of 1.25×10⁴ cells/well and serial dilutionsof different fusion proteins were added. The plates were then incubatedfor 48 h at 37° C. in a 5% CO2 atmosphere. Plates were developed byaddition of 20 μl of MTS solution (Promega) and analyzed at 490 nm usingan ELISA reader. Inhibition of proliferation (percent) was calculatedas: 100×[(ODexp−ODblank)/(ODmedium−ODblank)]×100.

Assay for Apoptosis

In brief, 1×10⁶ cells were treated with different fusion proteins for 72h. The cells were then washed with ice-cold PBS. The annexin V/propidiumiodide (PI) assay was conducted following procedures suggested by themanufacturer using the Vybrant Apoptosis Assay Kit 2 (Molecular Probes).

Proliferation of CFSE-Labeled 38C13/HER2 Tumor Cells

In brief, 1×10⁶ cells were incubated with 2.5 μM CFSE (Molecular Probes)for 10 min at 37° C. Cells were then treated with 1 nM of differentfusion proteins for 48 h and analyzed by flow cytometry followingprocedures suggested by the manufacturer using the CellTrace CFSE CellProliferation Kit (Molecular Probes).

Mice

Female C3H/HeN mice 6-8 wk of age obtained from Taconic Farms were used.Animals were housed in a facility using autoclaved polycarbonate cagescontaining wood-shaving bedding. The animals received food and water adlibitum. Artificial light was provided under a 12/12-h light/dark cycle.The temperature of the facility was 20° C. with 10-15 air exchanges perhour.

Half-Life

Murine rIFN-α (PBL Biomedical Laboratories), IgG3-IFN-α, andanti-HER2/neu-IgG3-IFN-α were iodinated to 10 μCi/μg with ¹²⁵I usingIodo-Beads (Pierce) according to the manufacturer's protocol. Mice wereinjected i.p. with 66 μCi of ¹²⁵I-labeled proteins. At various intervalsafter injection of ¹²⁵I-labeled rIFN-α, IgG3-IFN-α, oranti-HER2/neu-IgG3-IFN-α, residual radioactivity was measured using amouse whole body counter (Wm. B. Johnson and Associates).

Tumor Challenge and Ab Therapy

C3H/HeN mice received 1000 38C13/HER2 tumor cells s.c. Treatment wasgiven by i.p. injection either 1, 3, and 5 days or 12, 13, and 14 daysafter tumor challenge. Tumors were measured every other day, and thetumor volume (in cubic millimeters) was approximated using the followingformula: [length (mm)×width (mm)×width (mm)]/2 (Carlsson et al. (1983)J. Cancer Res. Clin. Oncol. 105: 20-23). Animals were observed until thelength of the s.c. tumor reached 15 mm or until any mouse was observedto be suffering or appeared to be moribund. Animals under theseconditions were euthanized humanely according to institutional policy.

Western Blot Analysis and Ab

In brief, 38C13/HER2 cells were treated with different fusion proteinsfor the indicated times, washed with ice-cold PBS, and lysed on ice for10 min in lysis buffer (0.125% Nonidet P-40, 0.875% Brij 97, 10 mMTris-HCl (pH 7.5), 2 mM EDTA, 0.15 M NaCl, 0.4 mM Na3VO4, 0.4 mM NaF, 1mM PMSF, 2.5 μM leupeptin, and 2.5 μM aprotinin) Cell lysates wereclarified at 10,000×g for 10 min at 4° C. Protein samples were thenboiled in sample buffer before separation on 8% SDS-PAGE gels andtransferred onto polyvinylidene fluoride microporous membranes(Millipore). After blocking with 3% BSA in 150 mM NaCl, 50 mM Tris-HCl(pH 7.6; TBS) for 1 h at room temperature, blots were probed with theindicated primary Abs overnight at 4° C. The blots were then washedthree times at room temperature with 0.05% Tween 20 in TBS, incubatedwith the appropriate secondary Abs conjugated with HRP, and detected bya peroxidase-catalyzed ECL detection system (ECL; Pierce). Polyclonalrabbit antiphosphoSTAT1 was obtained from Cell Signaling Technology.Polyclonal HRP-conjugated donkey anti-rabbit IgG was obtained fromAmersham Biosciences. Polyclonal rabbit anti-GAPDH was obtained fromAbcam.

Statistical Analysis

Statistical analyses were performed using a two-tailed Student's t testfor in vitro studies and log-rank (Mantel-Cox) analysis for animalsurvival curves.

Results

Production and Characterization of Anti-HER2/Neu-IgG3-IFN-α

The construction and expression of anti-HER2/neu-IgG3 with the C6 MH3-B1(20) variable region has been described previously (Huang and Morrison(2005) J. Pharmacol. Exp. Ther. 316: 983-991). The amino-terminal end ofmature murine IFN-α was fused to the carboxyl-terminal end ofanti-HER2/neu-IgG3 separated by a flexible [(Gly₄)Ser]₃ (SEQ ID NO:5)linker (FIG. 2A). An identical fusion protein, IgG3-IFN-α, lackingHER2/neu specificity was constructed by replacing the C6MH3-B1 L chainwith the 4D5 (rhuMab HER2, herceptin; Genentech) L chain. The proteinspurified from culture supernatants using protein G were analyzed bySDS-PAGE under nonreducing and reducing conditions (FIG. 2B). In theabsence of reducing agents, anti-HER2/neu-IgG3 (FIG. 2B, lane 1)migrates with a molecular mass of 170 kDa, whereasanti-HER2/neu-IgG3-IFN-α (FIG. 2B, lane 2) and IgG3-IFN-α (FIG. 2B, lane3) are 210 kDa, the size expected for a complete IgG3 with two moleculesof murine IFN-α attached (FIG. 2A). After treatment with the reducingagent, L chains migrating with a molecular mass of 25 kDa are seen forthese proteins (FIG. 2B, lanes 4-6). However, the anti-HER2/neu-IgG3 hasan H chain with a molecular mass of 60 kDa (FIG. 2B, lane 4), whereasIgG3-IFN-α (FIG. 2B, lane 5) and anti-HER2/neu-IgG3-IFN-α (FIG. 2B, lane6) have an H chain with a molecular mass of 80 kDa as expected. Thelower band in lane 1 (FIG. 2B) is bovine IgG which also bound to theprotein G column; the bovine H and L chains are also seen in lane 4(FIG. 2B) and to a lesser degree in lanes 5 and 6 (FIG. 2B). FPLCanalysis showed that the IgG3-IFN-α fusion protein existed as a monomerin solution (data not shown).

Ag Binding and Antiviral Activity of Anti-HER2/Neu-IgG3-IFN-α

Both anti-HER2/neu-IgG3 and anti-HER2/neu-IgG3-IFN-α bound CT26/HER2cells, which express high levels of human HER2/neu, while IgG3-IFN-αbound CT26/HER2 weakly (FIG. 2C). Many cytokines including IL-1, IL-2,IL-6 (Ramsden and Rider (1992) Eur. J. Immunol. 22: 3027-3031) and IFN-α(Fernandez-Botran et al. (1999) Cytokine 11: 313-325) have been shown tointeract with heparin. To determine whether the weak interaction betweenIgG3-IFN-α and CT26/HER2 is due to the heparin binding, proteins wereincubated with heparin before the addition to CT26/HER2. Heparininhibited the binding of IgG3-IFN-α to CT26/HER2 cells but did notinhibit the binding of anti-HER2/neu-IgG3 and anti-HER2/neu-IgG3-IFN-α(FIG. 2C).

These results demonstrated that anti-HER2/neu-IgG3-IFN-α retained itsability to bind Ag and IgG3-IFN-α does not recognize HER2/neu. The L-929fibroblast cell line sensitive to VSV infection was used to quantify theIFN-α biological activity of the fusion proteins in comparison to anIFN-α standard. Both anti-HER2/neu-IgG3-IFN-α and IgG3-IFN-α exhibited2400 U of IFN-α activity/μg activity against VSV-induced cytotoxicity inL-929 cells, while anti-HER2/neu-IgG3 exhibited no anti-viral activity(FIG. 2D).

In Vivo Antitumor Activity of Fusion Proteins

To determine the in vivo anti-tumor activity ofanti-HER2/neu-IgG3-IFN-α, syngeneic mice were inoculated s.c. with 1×10³38C13/HER2 tumor cells and treated on days 1, 3, and 5 after tumorchallenge by i.p. administration of different doses of protein (FIG.3A-3B). Mice treated with 2.5 μg of IgG3-IFN-α showed some regression oftumor growth, with one (13%) of eight mice alive after 50 days (FIG.3A). However, in vivo targeting of IFN-α to tumors using atumor-specific Ab dramatically improved its antitumor effect. All micetreated with 2.5 μg (FIG. 3A) of anti-HER2/neu-IgG3-IFN-α remained tumorfree 50 days after tumor challenge (p=0.0048 compared with PBS control),and none of the treated mice showed evidence of toxicity. Thus,targeting of IFN-α to the tumor cell surface resulted in significantantitumor activity compared with IFN-α linked to a nonspecific Ab(p=0.007). Targeted anti-HER2/neu-IgG3-IFN-α continued to show potentantitumor activity when a lower dose was used. Seven (88%) of eight micetreated with 1 μg (FIG. 3B) of anti-HER2/neu-IgG3-IFN-α remained tumorfree after 50 days. In marked contrast, at this lower dose mice treatedwith IgG3-IFN-α showed tumor growth similar to mice treated with PBS(p=0.183) and only one (13%) of eight mice survived. When the treatmentwas increased to three doses of 5 μg, both anti-HER2/neu-IgG3-IFN-α andIgG3-IFN-α were effective in preventing tumor growth (data not shown)undoubtedly reflecting the fact that 38C13 cells are sensitive to IFN-αtreatment (Reid et al. (1989) Cancer Res. 49: 4163-4169; Basham et al.(1986) J. Immunol. 137: 3019-3024; Basham et al. (1988) J. Immunol. 141:2855-2860). Tumor growth in mice treated with 5 μg of anti-HER2/neu-IgG3Ab was the same as the PBS control, suggesting that Ab alone has noantitumor effect in vivo (data not shown). These results indicated thattargeting of IFN-α to the tumor cells by a tumor-specific Ab candramatically potentiate its effectiveness which was most clearly seenwhen low doses were administered. Importantly, this antitumor activitycan be achieved without any evident toxicity.

IFN-α Fused to an Ab Results in Improved Antitumor Activity Comparedwith Free IFN-α

As described above, we found that IFN-α fused to a non-tumor specific Abexhibited antitumor activity. To compare its antitumor activity withthat of soluble rIFN-α, mice were inoculated s.c. with 1×10³ 38C13/HER2tumor cells and treated 1 and 3 days after tumor challenge by i.p.administration of 9600 U (4 μg) of IgG3-IFN-α or 9600 U of rIFN-α (FIG.4A). All mice treated with 9600 U of IgG3-IFN-α showed delayed tumorgrowth and 75% of the mice remained tumor free 50 days after tumorchallenge (p=0.027). In contrast, mice treated with the same number ofunits of rIFN-α were not statistically different from PBS controls intheir tumor growth pattern.

IFN-α has a very short in vivo half-life (Bailon et al. (2001)Bioconjugate Chem. 12: 195-202). In previous study, fusion of Abs tocytokines has been shown to increase their halflife (Dela Cruz et al.(2000) Immunol. 165: 5112-5121). The clearance of ¹²⁵I-labeled rIFN-α,IgG3-IFN-α, or anti-HER2/neu-IgG3-IFN-α was examined in C3H/HeN mice.Mice were injected i.p. with 66 μCi of ¹²⁵I-labeled proteins and theresidual radioactivity was measured using a mouse whole body counter.rIFN-α was cleared rapidly with 50% eliminated by ˜2.5 h (FIG. 4B). Incontrast, both anti-HER2/neu-IgG3-IFN-α and IgG3-IFN-α exhibitedsignificantly increased in vivo half-life with ˜8 h required forelimination of 50% of the injected radioactivity. This increasedhalf-life may contribute to the antitumor efficacy of the IFN-α fusionproteins. Thus, fusion of an IgG3 Ab to IFN-α can significantly improveits in vivo antitumor activity. However, this antitumor activity can befurther improved by targeting the IFN-α to the tumor, making iteffective at lower doses.

Anti-HER2/Neu-IgG3-IFN-α Inhibited Proliferation of Tumor Cells In Vitro

IFN-α has multiple activities including activation of the immuneresponse and direct cytotoxicity against tumors. To investigatepotential mechanisms of the antitumor effects seen using eitheranti-HER2/neu-IgG3-IFN-α or IgG3-IFN-α, the eight mice remaining tumorfree (see FIG. 3A) were challenged with 1×10³ 38C13/HER2 tumor cells.Surprisingly, all mice resembled untreated mice and quickly developedbulky tumors (data not shown). These results imply that under theseexperimental conditions of low tumor burden the IFN-α fusion proteinsdid not initiate a protective adaptive immune response, but instead thepotent antitumor activity of the IFN-α fusion proteins is mediatedeither by the innate immune system or by a direct cytotoxic effect ontumor cells.

To determine whether IFN-α fusion proteins are directly cytotoxic totumor cells, the 38C13/HER2 or parental 38C13 tumor cells were incubatedwith different proteins for 48 h and cell proliferation measured usingthe MTS assay. Treatment with anti-HER2/neu-IgG3 did not significantlyinhibit the proliferation of either 38C13/HER2 or parental 38C13 tumorcells (FIGS. 5A and 5B). Although both anti-HER2/neu-IgG3-IFN-α andIgG3-IFN-α inhibited the proliferation of 38C13/HER2 tumor cells,anti-HER2/neu-IgG3-IFN-α was more effective than IgG3-IFN-α with IPSOvalues of 10 and 100 pM for anti-HER2/neu-IgG3-IFN-α and IgG3-IFN-α,respectively (FIG. 5A). In contrast, anti-HER2/neu-IgG3-IFN-α andIgG3-IFN-α exhibited similar antiproliferative activity against parental38C13 tumor cells. These results provided evidence that IFN-α fusionproteins can directly inhibit the proliferation of the B cell lymphoma38C13, and targeting IFN-α to tumor cells potentiated this effect.

Anti-HER2/Neu-IgG3-IFN-α Induced Apoptosis in Tumor Cells In Vitro

IFN-α signaling can induce apoptosis in some tumor cell lines. Todetermine whether apoptosis contributed to the antiproliferative effectwe observed, 38C13/HER2 cells treated with different proteins wereassayed for the translocation of phosphatidylserine from the inner tothe outer leaflet of the plasma membrane using the annexin V-affinityassay (Koopman et al. (1994) Blood 84: 1415-1420). Dead cells werestained by PI, which enters cells with a disrupted plasma membrane andbinds to DNA. Compared with the PBS control, there was no increase inthe number of dead cells (annexin V/PI bright, 2%) or early apoptoticcells (annexin V bright, 3%) following treatment with anti-HER2/neu-IgG3(FIG. 5C). In contrast, when cells were treated with IgG₃-IFN-α, therewas a significant increase in the number of dead cells (21%) and earlyapoptotic cells (6%). Treatment with anti-HER2/neu-IgG3-IFN-α resultedin a further increase in both the number of dead cells (33%) and earlyapoptotic cells (16%). These results indicated that IFN-α can induceapoptosis in 38C13/HER2 tumor cells, and that targeting IFN-α to tumorcells can markedly increase this effect.

In addition to inducing apoptosis, IFN-α can directly inhibit theproliferation of tumor cells (Tiefenbrun et al. (1996) Mol. Cell. Biol.16: 3934-3944). To determine whether both inhibition of proliferationand apoptosis were taking place in treated tumor cells, CFSE-labeled38C13/HER2 cells were treated with different proteins for 48 h, the livecells were gated, and the level of CFSE was determined by flowcytometry. The CFSE signal in anti-HER2/neu-IgG3-treated cells (FIG. 5D,thin line) overlapped with the PBS-treated cells and was significantlyless than that of cells fixed immediately after CFSE labeling (FIG. 5D,dotted line), indicating that anti-HER2/neu-IgG3 did not inhibit theproliferation of the 38C13/HER2. In contrast, IgG3-IFN-α significantlyinhibited the proliferation of the surviving 38C13/HER2 cells (FIG. 5D,thick line), and targeting IFN-α to 38C13/HER2 cells byanti-HER2/neu-IgG3-IFN-α potentiated this effect (FIG. 5D, black area).These results indicated that although anti-HER2/neu-IgG3-IFN-α treatmentdid not result in complete cell death by 48 h, the surviving cells had areduced ability to proliferate.

IFN-α Fusion Proteins Induce STAT1 Activation in Tumor Cells

Although engagement of the IFN-α receptor can initiate activation ofmultiple STAT proteins, STAT1 plays an obligate role in mediatingIFN-α-dependent signaling (Meraz et al. (1996) Cell 84: 431-442). Toinvestigate whether IFN-α fusion proteins initiate IFN-α signaling in38C13/HER2 and that targeting IFN-α to tumor cells augments this effect,the phosphorylation of STAT1 following treatment was examined. As shownin FIG. 6A-6C, both anti-HER2/neu-IgG3-IFN-α and IgG3-IFN-α initiatedrobust STAT1 phosphorylation in 38C13/HER2 with STAT1 phosphorylationincreasing 8-fold by 10 min. However, the phosphorylation of STAT1induced by anti-HER2/neu-IgG3-IFN-α persisted for a longer period oftime and greater STAT1 phosphorylation was seen at 30, 60, and 90 min incells treated with anti-HER2/neu-IgG3-IFN-α. These results indicatedthat IFN-α fusion proteins can induce IFN-α signaling in 38C13 lymphomacells and targeting IFN-α to tumor cells augments this effect.

Anti-HER2/Neu-IgG3-IFN-α Exhibited Potent Activity Against EstablishedTumors

Because anti-HER2/neu-IgG3-IFN-α exhibited potent cytotoxicity against38C13/HER2 tumor cells, we investigated whether anti-HER2/neu-IgG3-IFN-αwould be effective against established 38C13/HER2 tumors. Syngeneic micewere inoculated s.c. with 1×10³ 38C13/HER2 tumor cells and i.p. treatedwith 5 μg (FIG. 7) of the indicated proteins on days 12, 13, and 14after tumor challenge. The average tumor size on day 12 is 100 mm³ andtreatment with PBS or 10 μg of anti-HER2/neu-IgG3 (data not shown) didnot inhibit tumor growth. Treatment with 5 μg of IgG3-IFN-α showed someeffect in inhibiting tumor growth; however, all mice developed bulkytumors and none of them survived 32 days after tumor challenge. Incontrast all mice treated with 5 μg of anti-HER2/neu-IgG3-IFN-α haddelayed tumor growth, and three of eight mice had complete tumorregression and remained tumor free 50 days after tumor challenge(anti-HER2/neu-IgG3-IFN-α vs PBS, p=0.0001; anti-HER2/neu-IgG3-IFN-α vsIgG3-IFN-α, p=0.063). Thus, both IgG3-IFN-α and anti-HER2/neu-IgG3-IFN-αshowed antitumor activity but anti-HER2/neu-IgG3-IFN-α was moreeffective in delaying tumor growth and complete tumor remission wasobserved only in mice treated with anti-HER2/neu-IgG3-IFN-α. When thetreatment dose was increased to 10 μg of the fusion proteins, almost allmice treated with either anti-HER2/neu-IgG3-IFN-α or IgG3-IFN-α hadcomplete tumor regression and remained tumor free after 50 days.

The mice that remained tumor free following treatment with three dosesof 10 μg of fusion proteins were rechallenged with 1×10³ 38C13/HER2tumor cells on day 50. All mice remained tumor free (data not shown).These results suggest that an adaptive immune response with immunologicmemory is initiated when larger, established tumors are treated withIFN-α fused to an Ab.

Discussion

Although rIFN-α has shown activity against B cell lymphoma and multiplemyeloma, inconsistent efficacy and systemic toxicity have limited itsusefulness (Oken (1992) Cancer 70: 946-948). The present workdemonstrates that fusing IFN-α to an Ab improves its efficacy againsttumors with further improvement seen when IFN-α is targeted to tumorcells by a tumor-specific Ab. This antitumor efficacy is seen withoutany apparent toxicity. These studies suggest that fusion of IFN-α withtumor-specific Ab may yield an effective biologic agent for thetreatment of B cell lymphoma.

To test the hypothesis that directing IFN-α to tumor sites with Ab wouldresult in improved efficacy, we chose a well-characterized murine B celllymphoma engineered to express a common TAA, HER2/neu, to which Abs areavailable. Anti-HER2/neu-IgG3-IFN-α appears to be more effective in thetreatment of the 38C13 B cell lymphoma than previously describedimmunotherapeutics, although in the present study a foreign Agintroduced by gene transduction was the target. Treatment with three 1μg doses of anti-HER2/neu-IgG3-IFN-α beginning 1 day after tumorchallenge appeared to be as effective in inhibiting tumor growth astreatment with 10 μg of anti-Id IgG1-IL-2 fusion protein for 5 daysbeginning 1 day after tumor challenge (Liu et al. (1998) Blood 92:2103-2112). In addition, anti-HER2/neu-IgG3-IFN-α was effective againstestablished tumors (FIG. 7) while anti-Id IgG1-IL-2 had little antitumoractivity when treatment was begun either 3 or 7 days after tumorchallenge (Id). The ability to cure established tumors also suggeststhat Ab-targeted IFN-α is a more powerful therapeutic agent than GM-CSF(Tao and Levy (1993) Nature 362: 755-758), CTLA-4 (Huang et al. (2000)Blood 96: 3663-3670), or CD40 ligand (Huang et al. (2004) Int. J. Cancer108: 696-703) fused to the Id Ag since none of these vaccine strategieswas effective against established tumors. Therefore, targeting IFN-α totumor cells appears to be a promising approach for treating B celllymphoma.

Targeting IFN-α to tumor cells with a tumor-specific Ab increases theantitumor efficacy of IFN-α. Anti-HER2/neu-IgG3-IFN-α is more effectivein inhibiting proliferation and inducing apoptosis (FIG. 5A-5D) in38C13/HER2 than IgG3-IFN-α and treatment with either 2.5 or 1 μg ofanti-HER2/neu-IgG3-IFN-α was more effective in inhibiting growth ofsmall tumors in vivo than the same doses of IgG3-IFN-α (FIGS. 3A and3B). These results suggest that the tumor-specific Ab directs IFN-α tothe tumor, thereby improving its therapeutic index with decreasedsystemic toxicity.

Remarkably, IgG3-IFN-α exhibits a more potent antitumor activity thanrIFN-α (FIG. 4A). Although rIFN-α is effective in treatment of a varietyof tumors (Gastl et al. (1985) Onkologie 8: 143-144; Atzpodien et al.(1991) Semin Oncol. 18: 108-112; Krown et al. (1992) J. Clin. Oncol. 10:1344-1351), prolonged treatment with high doses is required to seeeffective antitumor activity in part because of the very short half-lifeof the cytokine. In this study, we demonstrated that fusion of an IgG3Ab to IFN-α significantly increased its half-life (FIG. 4B), and thisincreased half-life may contribute to the increased in vivo antitumoractivity of the fusion protein (FIG. 4A). In addition, the Fc region ofthe IgG3-IFN-α may help to target IFN-α to the Fc receptors present on Blymphoma cells and consequently increase the antitumor activity.Therefore, fusion of IFN-α to an IgG3 Ab may provide multiple advantagesin improving the antitumor efficacy of IFN-α.

Although IFN-α has multiple activities, including activation of theimmune response, it appears that direct cytotoxicity plays an importantrole in the potent antitumor activity of anti-HER2/neu-IgG3-IFN-α. BothIFN-α fusion proteins exhibited apoptotic and antiproliferativeactivities against 38C13/HER2 with tumor targeting significantlyincreasing these effects (FIG. 5A-5D). Although the IFN-α fusionproteins were very effective in treating small tumors (FIGS. 3A and 3B),none of the survivors developed an immune response that protectedagainst second tumor challenge, suggesting that the direct cytotoxicityof the IFN-α fusion proteins was very effective in killing the tumorcells and that the adaptive immunity did not play a role when there wasa small tumor burden. Because 38C13 is an extremely malignant B lymphomacell line and mice injected with as few as 200 cells can develop bulkytumors within 20 days (Huang et al. (2000) Blood 96: 3663-3670), theIFN-α fusion proteins must be very effective in killing most of theinoculated tumor cells to result in long-term survivors. Multiplemechanisms, including down-regulation of NF-κB (Rath and Aggarwal (2001)J. Interferon Cytokine Res. 21: 523-528), induction of apoptosis byactivating caspase-3 (Yanase et al. (2000) J. Interferon Cytokine Res.20: 1121-1129), and up-regulation of both TRAIL and TRAIL receptors(Oshima et al. (2001) Cytokine 14: 193-201), have been shown to beinvolved in IFN-α-mediated cytotoxicity against tumor cells, and wewould expect these mechanisms to contribute to the direct cytotoxicityagainst tumor cells seen with Ab-IFN-α fusion proteins. Consistent withthis, we observed STAT1 activation following treatment of tumor cellswith the fusion proteins (FIG. 6A-6C).

Although IFN-α fusion proteins failed to initiate a memory immuneresponse when mice were treated beginning one day after tumorinoculation, IFN-α fusion proteins initiated an immune response thatprotected against second tumor challenge when mice were treatedbeginning 12 days after tumor inoculation. Therefore, IFN-α fusionproteins can activate protective adaptive immunity in the presence of asizable tumor burden. Because IFN-α is capable of activating adaptiveimmunity via stimulation of DC differentiation and maturation (Santiniet al. (2000) J. Exp. Med. 191: 1777-1788), it is possible that theestablished tumors provide more TAAs for DC activation in the presenceof IFN-α. In addition, the foreign Ag human HER2/neu may contribute tothe antitumor immunity by increasing the immunogenicity of the tumorcells in this model.

CD20, an Ag expressed by B cells, is expressed in most B cell lymphomas(Riley and Sliwkowski (2000) Semin. Oncol. 27: 17-24), and anti-CD20(rituximab, Genentech) is one of the most successful cancertherapeutics, having tremendous efficacy against lymphoma with littletoxicity (McLaughlin et al. (1998) J. Clin. Oncol. 16: 2825-2833).Although anti-HER2/neu IgG3-IFN-α is very effective against 38C13/HER2,HER2/neu is not normally expressed in lymphoma cells and therefore, itprobably has limited therapeutic application in the treatment oflymphoma but should be effective in the treatments of cancers thatexpress HER2/neu. In contrast, fusion of IFN-α to anti-CD20 is expectedto yield a fusion protein effective against lymphoma with even greaterantitumor activity by combining the antilymphoma activity of anti-CD20and the potent immunostimulatory and cytotoxic activity of IFN-α in oneprotein. Additionally, IFN-α may further up-regulate CD20 expression aswas seen in patients with B cell lymphoma following IFN-α treatment(Sivaraman et al. (2000) Cytokines Cell Mol. Ther. 6: 81-87). We arecurrently studying the effects of anti-CD20-IFN-α fusion proteins inmurine models of B cell lymphoma.

In summary, we have constructed and characterized a novel fusion proteinin which IFN-α was linked to an antibody recognizing a TAA. Our resultsindicate that fusion of IFN-α to a tumor-specific antibody candramatically increase the efficacy of IFN-α with antitumor activityobserved without any apparent toxicity. Remarkably, the Ab-IFN-α fusionprotein was effective against established tumors. Therefore, IFN (e.g.,IFN-α) fused to a tumor-specific antibody shows promise for thetreatment of B cell lymphoma.

Example 2 Anti-CD20-IFNα Fusion Proteins

Introduction

Out initials studies had indicated that a fusion protein withanti-HER2/neu joined to IFN-α was an effective therapeutic for thetreatment of HER2/neu expressing lymphoma. We sought to extend thesestudies to show that fusion of IFN-α with anti-CD20 would be aneffective therapeutic for treating CD20 expressing lymphomas. CD20 ispresent on virtually all lymphomas. However, it should be noted thatHER2/neu is expressed on many cancers and it would be expected that theanti-HER2/neu fusion protein would be effective in treating these. Inthe anti-CD20 fusion protein, we would expect the IFN-α in the fusionprotein to both exert a direct cytotoxic effect against the tumor cellsand to help elicit an anti-tumor immune response.

Produce Recombinant Antibodies Specific for CD20.

The variable regions for anti-CD20 (rituximab) were amplified and clonedinto expression vectors for the production of chimeric antibodies withhuman kappa light chains and gamma 3 heavy chains. Protein was producedand its ability to recognize CD20 examined using flow-cytometry and thehuman B-cell line Daudi. As shown in FIG. 8, the recombinant proteinbinds as well as rituximab a recombinant IgG1.

Produce Antibody Fusion Proteins with Human Interferon Joined toAntibodies Specific for CD20

a. Design of Fusion Protein

In our initial attempt to make a fusion protein we joined IFN-α to thecarboxy-terminus of the human IgG3 gene using a flexible glycine-serinelinker consisting of (Gly₄Ser)₃ (SEQ ID NO:5). The heavy chain is showndiagrammatically in FIG. 9.

After verifying that the fusion protein vector had the correctnucleotide sequence, it was transfected with the chimeric anti-CD20light chain into NS0 cells. Transfectants were screened by ELISA for theproduction of IgG. The clone giving the highest signal was expanded andfollowing sub-cloning was grown in roller bottles. Supernatants werethen passed through protein A Sepharose columns, and the bound proteinseluted and analyzed by SDS-PAGE both unreduced and following reduction(see, FIG. 10). Although the isolated protein was assembled into H₂L₂molecules, most of the isolated protein was smaller than expected.Following reduction, most of the heavy chains were smaller than expectedand ran at the same position as a gamma-3 heavy chain lacking a fusionprotein. It appeared that the interferon was being removed from thefusion protein by proteolysis. Western blot analysis using anti-Fc andanti-interferon, confirmed that both of the upper bands were heavychains, but only the largest contained interferon.

Flexible linkers can be a target of proteolytic cleavage. Therefore, weshortened the linker to only one copy of Gly₄Ser (SEQ ID NO:6). Thesevectors and vectors with the extended linker were transientlytransfected along with the appropriate light chain into HEK293T-cells.Cells were radiolabeled by growth in ³⁵S-methionine, immunoglobulinsprecipitated with protein A and analyzed by SDS-PAGE (FIG. 11). Whereascleavage of fusion proteins with extended linkers is readily apparent,cleavage does not take place when the linker consists of only oneGly₄Ser (SEQ ID NO:6). Therefore, the linker used to produce the fusionprotein is important and can influence its stability.

b. Recognition of CD20 by the Fusion Proteins

To determine if the fusion protein recognizes CD20, the human cell lineDaudi which expresses CD20 was incubated with RITUXAN®,anti-DNS/IgG3-hu-IFN-α or anti-CD20/IgG3-hu-IFN-α. Theanti-CD20/IgG3-hu-IFN-α bound better than RITUXAN® (FIG. 12). Theanti-DNS/IgG3-hu-IFN-α fusion also showed some binding, although lessthan either CD20 specific protein. We hypothesize that the binding ofthe anti-DNS/IgG3-hu-IFN-α and the enhanced binding ofanti-CD20/IgG3-hu-IFN-α compared to RITUXAN® is because the hu-IFN-αmoiety binds to IFN receptors expressed on the Daudi cells

The Timmerman laboratory has produced a transfectant of the murinelymphoma 38C13 that expresses human CD20. Both RITUXAN® andanti-CD20/IgG3-mu-IFN-α bound the transfectant. Anti-DNS/IgG3-mu-IFN-αshowed no binding (FIG. 13).

c. Anti-Viral Activity of the Fusion Proteins

To assess the anti-viral activity of the hu-IFN-α fusion proteins, HeLacells were seeded at 2×10⁵ cells/ml and treated with two-fold serialdilutions of fusion protein or Roferon (recombinant human interferon 2a)for 24 hrs. Cells were then infected with VSV (vesicular stomatitisvirus) at a concentration of 4000 pfu/100 μl. After 72 hrs, cells werestained with 0.1% crystal violet. Protection against viral infection wasdetermined either by quantitating the cells surviving the infection bystaining with 0.1% crystal violet and determining the amount of dye ineach well using a a spot densitometer of by counting the number ofplaques. In both assays the fusion protein had significant IFN-αactivity but was about 100-fold reduced in activity compared to Roferon.

Growth Inhibition and Killing of Daudi Lymphoma Cells with the FusionProteins.

Two methods were used to assess the growth inhibition/killing oflymphoma cells expressing CD20 by the fusion proteins. It should benoted that for these experiments a human cell line, Daudi, thatnaturally expresses CD20 was used. In the first approach Daudi cellswere incubated with various concentrations of IFN-α, antibody or fusionprotein for 72 hrs and growth inhibition assessed using the CellTiter 96AQueous cell proliferation assay (FIG. 14). Although showing less IFN-αactivity in the anti-viral assay, anti-CD20/IgG3-hu-IFN-α and Roferonshowed a similar ability to inhibit lymphoma growth suggesting thattargeting the IFN-α enhances its cytotoxic effect.Anti-CD20/IgG3+Roferon did not show enhanced activity compared toRoferon alone. Anti-DNS/IgG3-hIFN-α, RITUXAN® and anti-CD20/IgG3 onlyshowed some growth inhibition at the highest concentration used. Itshould be noted that fusion protein was more active than RITUXAN® inpreventing cell growth in this assay.

In the second approach, Daudi cells were incubated with variousconcentrations of IFN-α, antibody or fusion protein for 72 hrs and thenstained with Annexin V and propidium iodide (PI) and analyzed by FLOWcytometry. Shown in FIG. 15 are the results obtained when 10 pM of thevarious proteins was used. Cells in the early phases of apoptosis areAnnexin V⁺P⁻; late apoptotic and dead cells are Annexin V⁺PI⁺.

These experiments demonstrate several things. RITUXAN® andanti-CD20/IgG3 both induce little to no apoptosis, even at the highestconcentrations tested. As would be expected, murine IFN-α is lesseffective against the human cell line than is human recombinant IFN-α(Roferon) and anti-DNS/IgG3-mIFN α which would not target the tumorcells is approximately as effective as recombinant murine IFN-α.However, targeting murine IFN-α to tumor cells using anti-CD20/IgG3-mIFNα results in effective induction of cell death. Anti-CD20/IgG3-hIFNα ismore effective than anti-DNS/IgG3-hIFN α, again demonstrating thecontribution of cell targeting to cell killing. In this in vitro assay,Roferon and anti-CD20/IgG3-hIFNα exhibit similar activity causing celldeath at concentrations as low as 1 pM (data not shown). However, itshould be pointed out that in vivo CD20/IgG3-hIFNα will target andaccumulate at the site of the tumor while Roferon will exhibit itsactivity throughout the body.

Growth Inhibition and Killing of 38C13-CD20 Lymphoma Cells with theFusion Proteins

As briefly mentioned above, the laboratory of Dr. John Timmerman hasdeveloped a murine lymphoma, 38C13-CD20, that expresses human CD20 andwill grow in syngenic C3H/HeJ mice. The availability of this cell linemakes it possible to examine the in vivo efficacy of our fusionproteins. 38C13-CD20 cells were incubated for 48 hours with variousantibodies and fusion proteins. Killing and apoptosis were thendetermined by staining cells with Annexin V and PI and examining themusing FLOW cytometry. When proteins were used at a concentration of 100μM (data not shown) both recombinant mIFN-αand anti-CD20-IgG3-mIFN-αwere very effective in causing apoptosis, with anti-CD20-IgG3-mIFN-αsomewhat more effective that recombinant mIFN-α. Some apoptosis wasinduced by treating 38C13-CD20 cells with anti-DNS-IgG3-mIFN-α orRITUXAN®. Treatment with anti-CD20/IgG3 at this concentration had noeffect on cell viability. When the treatment concentration was loweredto 10 pM (FIG. 16), recombinant mIFN-α and anti-CD20/IgG3-mIFN-αcontinued to be effective in causing apoptosis, withanti-CD20/IgG3-mIFN-α more effective that recombinant mIFN-α. Only asmall amount of apoptosis was seen following treatment withanti-DNS-IgG3-mIFN-α indicating that targeting of IFN-α usinganti-CD20-IgG3-mIFN-α resulted in a more effective therapeutic agent. Atthis concentration RITUXAN® caused little apoptosis, indicating thesuperiority of the anti-CD20-IgG3/mIFN-α fusion protein over the unfusedanti-CD20 antibody. Again, treatment with anti-CD20/IgG3 had no effecton cell viability. At a treatment dose of 1 pM, onlyanti-CD20-IgG3-mIFN-α induced apoptosis in 38C13-CD20 (data not shown).At a dose of 0.1 pM, none of the treatments induced apoptosis (data notshown).

As an alternative approach, 38C13-CD20 cells were treated with thevarious proteins at different concentrations and inhibition of growthmonitored using the MTS assay (FIG. 17). Anti-CD20/IgG3-mIFN-α was mosteffective in inhibiting cell growth, followed by recombinant mIFN-α.Some growth inhibition was observed with anti-DNS/IgG3-mIFN-α.Anti-CD20/IgG3 and RITUXAN® had little effect on cell growth. Thus, theresults obtained in this assay mirrored what was observed when apoptosiswas monitored.

Production and Characterization of Additional IgG-IFNα Fusion Proteins

a. Anti-CD20-IgG1-mIFNα and Anti-CD20-IgG1-hIFNα

The initial proteins were made with IFN-α fused to a human IgG3backbone. RITUXAN® is an IgG1. To determine if the immunoglobulinbackbone influenced the properties of the fusion proteins, fusionproteins with m-IFN-α and hu-IFN-α fused to IgG1 have now been produced.They were of the expected molecular weight.

Anti-CD20/IgG1-mIFNα was evaluated for its ability to induce apoptosisof 38C13-CD20 (FIG. 18). The studies showed it to be effective, possiblyeven more effective than the IgG3 fusion protein.

Anti-CD20/IgG1-hIFNα was evaluated for its ability to induce apoptosisof Daudi cells. The studies showed it exhibits activity similar toanti-CD20/IgG3-hIFNα (FIG. 19).

The fusion proteins were evaluated for their ability to inhibit thegrowth of Daudi cells as shown in FIG. 20. IgG1 fusions with both murineand human IFNα resembled the IgG3 fusions in their ability to inhibitthe growth of Daudi cells.

b. Fusion Proteins with IFN-α Joined to the IgG Backbone with an AlphaHelical Linker.

Fusion proteins were produced in which the GlySer linker was replacedwith linker with the sequence A(EAAAK)₂A (SEQ ID NO:7). This sequence isproposed to fold as an alpha helix.

Protein was produced by transient expression in 293T cells and evaluatedby SDS-PAGE. The protein assembled and was of the expected molecularweight. No cleavage of the linker was observed.

The fusion protein, anti-CD20-IgG3-hIFNα (α-helical linker) when used atthe same concentration as the fusion protein with the Gly₄Ser (SEQ IDNO:6) linker, was found to effectively induce apoptosis of Daudi cells(FIG. 21).

In Vivo Treatment of Tumors

The 38C13 lymphoma that had been transduced by the Timmerman laboratoryto express human CD20 was used for these studies. 38C13 is an aggressivelymphoma that grows in syngenic C3H/HeJ mice. The transductant,38C13-CD20, exhibits the same growth characteristic. Thus it is possibleto investigate fusion protein mediated protection in immune competentanimals.

a. Treatment of Early Tumors

Mice (groups of 4) were injected subcutaneously with 5000 38C13-CD20cells on day zero. On days 1, 2 and 3 they were treated intravenouslywith herpes buffered saline solution (HBSS) or 0.4 μg, 2 μg, or 10 μg ofanti-CD20-m-IFN-α and tumor growth monitored. By day 20 all of theanimals treated with HBSS had large tumors and had to be sacrificed. Incontrast, no tumor growth was seen in animals treated with 10 μg of thefusion protein; after day 20 tumors began to grow in 3 of the fouranimals treated with 0.4 μg of the fusion protein and 1 of the micetreated with 2 μg. The results showed that the anti-CD20/IFN-α fusionproteins are very effective in inhibiting in vivo tumor growth and inincreasing survival (see, e.g., FIG. 22).

b. The Anti-CD20-mIFNα Fusion Protein is More Effective than EitherRituximab or Anti-CD20/IgG3 in Treating Moderate Sized Tumors

C3H/HeJ mice were inoculated with 5000 38C13-CD20 cells on day 0. Ondays 5, 6 and 7 they were treated with HBSS or 10 μg of anti-CD20-IgG1(produced in 293T cells), anti-CD20-IgG3, rituximab oranti-CD20-IgG3-mIFNα. They were monitored for tumor growth and survival(see, e.g., FIG. 23). Anti-CD20/IgG3-mIFNα was much more effective thanrituximab, anti-CD20/IgG3 or anti-CD20/IgG1 in preventing the growth ofmoderate sized tumors.

The Tumor Targeting Ability of the Fusion Protein Significantly Enhancesits Efficacy in Vivo.

C3H/H3J mice were inoculated with 5000 38C13-CD20 cells on day 0 andtreated on days 5, 6 and 7 with 10 μg of anti-CD20-IgG3, 10 μg ofanti-CD20-IgG3+mIFN-α (dose chosen to be same moles as in fusionprotein), anti-DNS-IgG3-IFNα, or anti-CD20-IgG3-mIFNα and followed fortumor growth and survival (see, e.g., FIG. 24). Anti-CD20-IgG3-IFNαsignificantly delayed tumor growth and promoted survival indicating thattargeting the IFNα to the tumor using the antibody combining site makesit a more effective therapeutic than either a fusion protein that doesnot target the fused IFNα (anti-DNS-IgG3-IFNα) or the injection ofanti-CD20 along with IFNα that is not covalently associated(anti-CD20-IgG3+mIFN-α).

Fusion Protein Treatment is Effective Against Established Tumors

Groups of eight C3H/HeJ mice were inoculated with 5000 38C13-CD20 cellsand treated on days 8, 9 and 10 with 100 μg of anti-CD20-mIFNα or HBSS.Mice were monitored for tumor growth (see FIG. 25) and survival (see,FIG. 26). Mice inoculated with anti-CD20-mIFNα shows improved survival(FIG. 26).

Repeat Treatment with Fusion Protein Leads to Improved Efficacy

In the initial experiments, mice were treated with a single round ofinjections that significantly delayed tumor onset and enhanced survival.However, some animals eventually developed tumors. To determine ifrepeated dosing with anti-CD20-mIFNα could completely prevent tumorgrowth mice were treated with two additional doses of 30 μg of fusionprotein twelve and nineteen days following the initial treatments. Asshown in FIG. 27. Following the two additional treatments, 87% of theanimals were tumor free after 60 days indicating that they had beencured of their tumor. Importantly, there was no evidence of IFN-mediatedtoxicity in the treated animals whose normal cells express the murineIFNR. These results suggest that with an optimized treatment schedule,38C13-huCD20 tumor growth can be completely prevented and thatappropriate application of the fusion protein in the clinic can cureclinical disease.

Targeting IFNα Results in an Improved Anti-Tumor Activity

To quantify the IFNα activity of the fusion protein, MTS assay measuringcell viability were performed on the non-CD20 expressing parental 38C13cells. Using 38C13 cells, anti-CD20-IFNα and anti-DNS-mIFNα hadequivalent ability to inhibit the proliferation of non-CD20 expressing38C13 (FIG. 28). However, their activities were about 300-fold reducedcompared to recombinant mIFNα. In contrast anti-CD20-mIFNα had 105-foldhigher anti-proliferative activity than non-targeted controlanti-DNS-mIFNα against 38C13 cells that expressed CD20 (38C13-CD20)indicating that targeting of IFNα significantly enhances its efficacy.Compared to recombinant murine IFNα, anti-CD20-mIFNα had 103-fold higheranti-proliferative activity against 38C13-CD20. These experimentsillustrate two important points. First the fusion protein has reducedIFN activity which would be expected to decrease its toxicity. Secondly,targeting by the fusion protein enhances its activity so that it is morepotent that recombinant IFNα in inhibiting cell proliferation.

IFNAR Expression is Required for Anti-Tumor Activity of Anti-CD20-mIFNαActivity

IFNα has potent immunostimulatory and antitumor activities. It can acton tumor cells directly by inducing apoptosis upon binding to itsreceptor IFNAR on the cell surface, or indirectly by recruiting hostimmune cells such as NK cells into the tumor microenvironment to promotetumor killing. To distinguish between these possibilities, we used anshRNA approach to generate 38C13-huCD20 IFNAR KD, a cell line withdecreased expression of IFNAR (MFI=11) compared to its parent38C13-huCD20 (MFI=20) (FIG. 29A). Knockdown of IFNAR did not affect CD20expression as determined by flow cytometry (data not shown). In vitroapoptosis studies showed that 38C13-huCD20 IFNAR KD had a decreasedsensitivity to fusion protein treatment. At 48 hours post-treatment with1000 pM of anti-CD20-mIFNα, 57% of parental 38C13-huCD20 cells wereapoptotic compared to only 13% of the 38C13-huCD20 IFNAR KD cells (FIG.29B). Strikingly, in animal studies, the treatment regimen which hadpreviously been effective against 38C13-huCD20 failed to delay orprevent tumor onset in mice inoculated with 38C13-huCD20 IFNAR. The invivo growth kinetics of 38C13-huCD20 IFNAR KD were similar to those ofthe parental cell line 38C13-huCD20 (data not shown). Thus, IFNARexpression is required for anti-CD20-mIFNα-mediated activity in vivo.Data from these in vivo studies suggest that the anti-tumor effect ofanti-CD20-mIFNα is mediated primarily and possibly exclusively throughthe induction of tumor cell death via a direct interaction betweentargeted IFNα and its receptor present on the surface of tumor cells.

Anti-CD20-hIFNα is Active Against Human Cells and Completely CuresEstablished Human Xenograft Tumors.

In this example, the effect of anti-CD20-hIFNα on human xenograftstumors was investigated. Five to seven mice per group were inoculatedsubcutaneously with Daudi cells and treated as indicated in FIG. 35 withthree weekly doses of 30 μg fusion protein, the equivalent molarconcentration of rituximab, or HBSS. Treatment was administered 30, 37and 44 days post tumor inoculation (arrows) to mice with tumors at least0.5 cm in diameter. HBSS was injected as a control. Symbols representindividual mice. Panel D: Survival curves for the mice whose tumorgrowth is shown in panels A-C. *P=0.02.

When activity was evaluated using the human lymphoma Daudi, consistentwith what had been observed in the murine tumor model, anti-CD20-hIFNαhad far higher proapoptotic activity than rituximab or the combinationof rituximab and hIFNα. Importantly, the fusion protein was effective atvery low doses where rituximab treatment did not induce significantlevels of apoptosis.

One problem with rituximab treatment is that a subset of treatedpatients become refractory to treatment. A goal is to find an effectivetreatment for this patient population. rituximab resistant (RR)resistant clones of the human B cell lymphoma Ramos have been isolatedby growth in stepwise increasing concentrations of rituximab for 10 weekafter which single cell clones were isolated (Jazirehi et al. (2007)Cancer Res. 67:1270). Increased expression of Bcl-2, Bcl-xL, Mcl-1 andhyperactivation of the NF-kB and ERK1/2 pathways was seen in the RRclones. We have obtained these cells and show that RR1 is more sensitiveto treatment with anti-CD20hIFNα than is Ramos (FIG. 31). Thus,anti-CD20hIFNα holds promise for the treatment of rituximab resistantpatients although it is not certain that the rituximab resistance seenin the patients results from the same changes as those seen in thecultured cells.

Example 3 Evaluation of Targeted Interferon-β

All type I IFNs are recognized by a single shared receptor composed oftwo transmembrane proteins IFNAR1 and IFNAR2. At the level of receptorrecruitment, a prominent feature of IFN-β compared to IFN-α2 is astronger binding to the receptor. The half-life of the complex withIFNAR2-EC is about 20-fold higher for IFN-β compared to IFN-α2 and theaffinity of IFNAR1-EC for IFN-β is two orders of magnitude higher thanfor IFN-α2. IFN-α2 and IFN-β have very similar anti-viral activity, butdiffer significantly in their antiproliferative activity, with IFN-βbeing significantly more potent. Like IFN-α, IFN-β shows activityagainst malignancies. IFN-β has frequently been found to be moreeffective than IFN-α against non-hematopoietic tumors such as melanoma.Because of these differences, especially the higher affinity of IFN-βfor the IFN-receptor, we have now evaluated the efficacy ofanti-CD20-IFN-β fusion proteins.

The data showed that fusion proteins with murine IFN-β are extremelyeffective in inhibiting lymphoma proliferation (FIG. 32). The untargetedanti-DNS-mIFN-β is more active than mIFN-β on a molar basis, although itshould be noted that there are two molecules of IFN-β per mole of fusionprotein. As is seen with the IFN-α fusion proteins, targeting the fusionprotein to the antigen CD20 expressed on the surface of the lymphomamakes it even more potent. Comparison of the IFN-β fusion proteins withthe IFN-α fusion proteins showed that the IFN-β fusion proteins are evenmore potent.

Anti-CD20hIFN-β is Effective in Preventing Growth of Human LymphomaCells.

The antibody fusion proteins are effective in preventing theproliferation of Daudi cell. Daudi expressed human CD20 andanti-CD20hIFN-β is more effective than anti-DNShIFN-β showing thattargeting to antigens expressed on the surface of the lymphoma cellsmakes the fusion protein more potent (FIG. 33). In contrast to what wasseen with the murine IFN-β, the fusion proteins are not as active asrecombinant human IFN-β.

Anti-CD20-mIFNβ is Effective Against Cells Expressing Low Levels of theIFN Receptor.

As shown in FIG. 34 and Table 5, anti-CD20-mIFNβ is effective againstcells expressing low levels of the IFN receptor.

TABLE 5 Efficacy of fusion proteins against cells expressing low levelsof the interferon receptor. Anti- Anti- Anti- CD20- DNS- CD20- Anti-DNS-mIFN-β mIFN-β mIFN-α mIFN-α IC₅₀ (pM) 38C13-CD20 2.2 16.12 76.4 409.3IC₅₀ (pM) 38C13-CD20 - 10.1 102.0 848.4 Not IFNR Knock Down calculatedIC₅₀ (pM) of 38C13-CD20 and 38C13-CD20 IFNR Knock-Down cells treatedwith the indicated proteins.

In addition, anti-CD20-mIFNβ causes apoptosis in 38C13CD20 cells inwhich the level of IFN receptor expression has been decreased usingshRNA. Targeted anti-CD20-mIFNβ is more effective than non-targetedanti-DNS-mIFNβ or recombinant mIFNβ at similar concentrations

These studies show that targeted anti-CD20-mIFNβ is effective againstcells that express only low levels of the IFNR. Targeted anti-CD20-mIFNαwas not effective against these cells. This is consistent with thehigher affinity of IFNβ for the IFNR and suggests that fusion proteinswith IFNβ may be effective against cells that do not respond to IFNαtreatment.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A chimeric construct comprising a mutantinterferon attached to an antibody that binds to a tumor-associatedantigen, wherein: said antibody is a full-length antibody that binds atumor-associated antigen; said antibody is attached to said mutantinterferon by a peptide linker, the amino acid sequence of said linkerconsisting of the sequence SGGGGS (SEQ ID NO:81) or AEAAAKEAAAKAGS (SEQID NO:82); said mutant interferon is a mutant interferon alpha where themutations in said mutant interferon consist of one or more mutationsselected from the group consisting of H57Y, E58N, and Q61S wherein theposition is relative to the human wildtype interferon α2; and whereinthe construct when contacted to a tumor cell results in the killing orthe inhibition of the growth or proliferation of the tumor cell.
 2. Thechimeric construct of claim 1, wherein said antibody is an anti-CD20antibody.
 3. The chimeric construct of claim 1, wherein said antibody isan anti-HER2 antibody.
 4. The chimeric construct of claim 1, whereinsaid antibody is an anti-CD33 antibody.
 5. The chimeric construct ofclaim 2, wherein the mutant interferon comprises mutations H57Y, E58N,and Q61S.
 6. The chimeric construct of claim 3, wherein the mutantinterferon comprises mutations H57Y, E58N, and Q61S.
 7. The chimericconstruct of claim 4, wherein the mutant interferon comprises mutationsH57Y, E58N, and Q61S.
 8. The chimeric construct of claim 1, wherein thechimeric construct has reduced anti-proliferative activity against cellslacking expression of said tumor-associated antigen compared to themutant interferon not attached to an antibody that binds thetumor-associated antigen.
 9. The chimeric construct of claim 1, whereinthe chimeric construct has enhanced anti-proliferative activity againstcells expressing said tumor-associated antigen compared to the mutantinterferon not attached to an antibody that binds the tumor-associatedantigen.
 10. The chimeric construct of claim 1, wherein the mutantinterferon retains at least 80% biological activity of the wildtypeinterferon.
 11. The chimeric construct of claim 1, wherein the antibodyis an antibody that specifically binds to tumor-associated antigenselected from the group consisting of CD20, HER3, HER2/neu, mucin 1(MUC-1), G250, CD33, mesothelin, gp100, tyrosinase, andmelanoma-associated antigen (MAGE).